Cervical carcinoma is one of the most common malignant diseases world-wide and is one of the leading causes of morbidity and mortality among women (Parkin D M, Pisani P, Ferlay J (1993) Int J Cancer 54: 594-606; Pisani P, Parkin D M, Ferlay J (1993) Int J Cancer 55: 891-903). 15,700 new cases of invasive cervical cancer were predicted in the United States in 1996, and the annual world-wide incidence is estimated to be 450,000 by the World Health Organization (1990). The annual incidence rate differs in different parts of the world, ranging from 7.6 per 100,000 in western Asia to 46.8 per 100,000 in southern Africa (Parkin et al., 1993 ibid).
The current conception of cervical carcinoma is that it is a multistage disease, often developing over a period of 10-25 years. Invasive squamous-cell carcinoma of the cervix is represented by penetration through the basal lamina and invading the stroma or epithelial lamina propria. The clinical course of cervical carcinoma shows considerable variation. Prognosis has been related to clinical stage, lymph node involvement, primary tumour mass, histology type, depth of invasion and lymphatic permeation (Delgado G, et al., (1990) Gynecol Oncol 38: 352-357). Some patients with less favourable tumour characteristics have a relatively good outcome, while others suffer a fatal outcome of an initially limited disease. This shows a clear need for additional markers to further characterise newly diagnosed cervical carcinomas, in order to administer risk-adapted therapy (Ikenberg H, et al., Int. J. Cancer 59:322-6. 1994).
The epidemiology of cervical cancer has shown strong association with religious, marital and sexual patterns. Almost 100 case-control studies have examined the relationship between HPV and cervical neoplasia and almost all have found positive associations (IARC monographs, 1995). The association is strong, consistent and specific to a limited number of viral types (Munoz N, Bosch F X (1992) HPV and cervical neoplasia: review of case-control and cohort studies. IARC Sci Publ 251-261). Among the most informative studies, strong associations with HPV 16 DNA have been observed with remarkable consistency for invasive cancer and high-grade CIN lesions, ruling out the possibility that this association can be explained by chance, bias or confounding (IARC monographs, 1995). Indirect evidence suggested that HPV DNA detected in cancer cells is a good marker for the role of HPV infection earlier in the carcinogenesis. Dose-response relationship has been reported between increasing viral load and risk of cervical carcinoma (Munoz and Bosch, 1992 ibid). In some larger series up to 100% of the tumours were positive for HPV but the existence of virus-negative cervical carcinomas is still debatable (Meijer C J, et al., (1992) Detection of human papillomavirus in cervical scrapes by the polymerase chain reaction in relation to cytology: possible implications for cervical cancer screening. IARC Sci Publ 271-281; Das B C, et al., (1993) Cancer 72: 147-153).
The most frequent HPV types found in squamous-cell cervical carcinomas are HPV 16 (41%-86%) and 18 (2%-22%). In addition HPV 31, 33, 35, 39, 45, 51, 52, 54, 56, 58, 59, 61, 66 and 68 are also found (IARC, monographs, 1995). In the HPV2000 International conference in Barcelona HPV 16, 18, 31 and 45 were defined as high risk, while HPV 33, 35, 39, 51, 52, 56, 58, 59, 68 were defined as intermediate risk (Keerti V. Shah. P71). The 13 high risk plus intermediate risk HPVs are together often referred to as cancer-associated HPV types.
A number of studies have explored the potential role of HPV testing in cervical screening (see Cuzick et al. A systematic review of the role of human papillomavirus testing within a cervical screening programme. Health Technol Assess 3:14. 1999).
Reid et al., (Reid R, et al., (1991) Am J Obstet Gynecol 164: 1461-1469) where the first to demonstrate a role for HPV testing in a screening context. This study was carried out on high-risk women from sexually transmitted disease clinics and specialist gynaecologists, and used a sensitive (low stringency) Southern blot hybridisation for HPV detection. A total of 1012 women were enrolled, and cervicography was also considered as a possible adjunct to cytology. Twenty-three CIN II/III lesions were found altogether, but only 12 were detected by cytology (sensitivity 52%, specificity 92%). HPV testing found 16 high-grade lesions.
Bauer et al. (Bauer H M, et al., (1991) JAMA 265: 472-477) report an early PCR-based study using MY09/11 primers (Manos M, et al., (1990) Lancet 335: 734) in young women attending for routine smears (college students). They found a positive rate of 46% in 467 women, which was much higher than for dot blot assay (11%).
In a study using PCR with GP5/6 primers (Van Den Brule A J, et al., (1990) J Clin Microbiol 28: 2739-2743) van der Brule et al. (Van Den Brule A J, et al., (1991) Int J Cancer 48: 404-408) showed a very strong correlation of HPV positivity with cervical neoplasia as assessed by cytology. In older women (aged 35-55 years) with negative cytology the HPV positivity rate was only 3.5%, and this was reduced to 1.5% if only types 16, 18, 31 and 33 were considered, while women with histological carcinoma in situ were all HPV-positive, and 90% had one of the four above types. Women with less severe cytological abnormalities had lower HPV positivity rates in a graded way, showing a clear trend.
Roda Housman et al. (Roda Housman A M, et al., (1994) Int J Cancer 56: 802-806) expanded these observations by looking at a further 1373 women with abnormal smears. This study also confirmed increasing positivity rate with increasing severity of smear results. They also noted that the level of HPV heterogeneity decreased from 22 types for low-grade smears to ten “high-risk” types for high grade smears. This paper did not include any cytologically negative women, nor was cytological disease confirmed histologically.
Cuzick et al. (Cuzick J, et al., (1992) Lancet 340: 112-113; Cuzick J, et al., (1994) Br J Cancer 69: 167-171) were the first to report that HPV testing provided useful information for the triage of cytological abnormalities detected during random screening. In a study of 133 women, referral for coloposcopy they found a positive predictive value of 42%, which was similar to that for moderate dyskaryosis. The results were most striking for HPV 16, where 39 of 42 HPV 16 positive women were found to have high-grade CIN on biopsy. This study pointed out the importance of assessing viral load and only considered high levels of high-risk types as positive.
Cox et al. (Cox J T, et al., (1995) Am J Obstet Gynecol 172: 946-954) demonstrated a role for HPV testing using the Hybrid Capture™ system (DIGENE Corporation, Gaithersburg, Md., USA) for triaging women with borderline smears. This test was performed on 217 such women from a college referral service, and a sensitivity of 93% was found for CINII/III compared with 73% for repeat cytology. High viral load was found to further improve performance by reducing false positives. When 5 RLU was taken as a cut-off, a PPV of approximately 24% was found with no loss of sensitivity.
Cuzick et al. (Cuzick J, et al., (1995) Lancet 345: 1533-1536) evaluated HPV testing in a primary screening context in 1985 women attending for routine screening at a family planning clinic. Sensitivity using type-specific PCR for the four common HPV types (75%) exceeded that of cytology (46%), and the PPV for a positive HPV test (42%) was similar to that for moderate dyskaryosis (43%).
WO 91/08312 describes methods for determining the prognosis of individuals infected with HPV which comprise measuring the level of HPV activity by detecting transcripts of all or a portion of the E6 and/or E7 HPV genes in a sample and comparing the measurements of HPV activity with a previously established relationship between activity and risk of progression to serious cervical dysplasia or carcinoma.
WO 99/29890 describes methods for the assessment of HPV infection based on the measurement and analysis of gene expression levels. In particular, WO 99/29890 describes methods which are based on measuring the levels of expression of two or more HPV genes (e.g. HPV E6, E7, L1 and E2) and then comparing the ratio of expression of combinations of these genes to provide an indication of the stage of HPV-based disease in a patient.
The present inventors have determined that it is possible to make a clinically useful assessment of HPV-associated disease based only on a simple positive/negative determination of expression of HPV L1 and E6 mRNA transcripts, with no requirement for accurate quantitative measurements of expression levels or for determination of differences in the levels of expression of the two transcripts. This method is technically simple and, in a preferred embodiment, is amenable to automation in a mid-to-high throughput format. Furthermore, on the basis of results obtained using the method of the invention the inventors have defined a novel scheme for classification of patients on the basis of risk of developing cervical carcinoma which is related to disease-relevant molecular changes in the pattern of HPV gene expression and is independent of CIN classification.
Therefore, in a first aspect the invention provides an in vitro method of screening human subjects to assess their risk of developing cervical carcinoma which comprises screening for expression of mRNA transcripts from the L1 gene and the E6 gene of human papillomavirus, wherein subjects positive for expression of L1 and/or full length E6 mRNA are scored as being at risk of developing cervical carcinoma.
A positive screening result in the method of the invention is indicated by positive expression of L1 mRNA and/or E6 mRNA in cells of the cervix. Positive expression of either one of these mRNAs or both mRNAs is taken as an indication that the subject is “at risk” for development of cervical carcinoma. Women who express E6 mRNA are at high risk of developing cell changes because oncogenic E6 and E7 bind to cell cycle regulatory proteins and act as a switch for cell proliferation. Clear expression of E6 mRNA provides a direct indication of cell changes in the cervix. Expression of L1 mRNA, with or without expression of E6 mRNA is also indicative of the presence of an active HPV.
In the wider context of cervical screening, women identified as positive for L1 and/or E6 mRNA expression may be selected for further investigation, for example using cytology. Thus, at one level the method of the invention may provide a technical simple means of pre-screening a population of women in order to identify HPV-positive subjects who may be selected for further investigation.
In a specific embodiment, the method of the invention may be used to classify subjects into four different classes of risk for developing cervical carcinoma on the basis of positive/negative scoring of expression of L1 and E6 mRNA.
Accordingly, in a further aspect the invention provides an in vitro method of screening human subjects to assess their risk of developing cervical carcinoma which comprises screening the subject for expression of mRNA transcripts of the L1 gene of HPV and mRNA transcripts of the E6 gene of HPV, and sorting the subject into one of four categories of risk for development of cervical carcinoma based on expression of L1 and/or E6 mRNA according to the following classification:
Risk category 1: subjects negative for expression of L1 mRNA but positive for expression of E6 mRNA from at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 or 68. Those individuals positive for expression of E6 mRNA from at least one of HPV types 16, 18, 31 or 33 are scored as being at higher risk, for example in comparison to individuals negative for these types but positive for expression of E6 mRNA from at least one of HPV types 35, 39, 45, 52, 56, 58, 59, 66 or 68.Risk category 2: subjects positive for expression of L1 mRNA and positive for expression of E6 mRNA from at least one of HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 or 68. Those individuals positive for expression of E6 mRNA from at least one of HPV types 16, 18, 31 or 33 are scored as being at higher risk, for example in comparison to individuals negative for these types but positive for expression of E6 mRNA from at least one of HPV types 35, 39, 45, 52, 56, 58, 59, 66 or 68.Risk category 3: subjects positive for expression of L1 mRNA but negative for expression of E6 mRNA from the cancer-associated HPV types, (e.g. negative for expression of E6 mRNA from HPV types 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66 and 68).Risk category 4: subjects negative for expression of L1 mRNA and negative for expression of E6 mRNA.
In a preferred embodiment, positive expression is indicated by the presence of more than 50 copies of the transcript per ml (or total volume of the sample) and negative expression is indicated by the presence of less than 1 copy of the transcript per ml (or total volume of the sample).
The above classification is based on molecular events which are relevant to risk of developing cervical carcinoma and is independent of the CIN status of the subjects. Thus, this method of classification may provide an alternative to the use of cytology in the routine screening of women to identify those at potential risk of developing cervical carcinoma. The method may also be used as an adjunct to cytology, for example as a confirmatory test to confirm a risk assessment made on the basis of cytology.
Women positive for expression of high risk E6 mRNA from one of HPV types 16, 18, 31 or 33 but negative for expression of L1 are in the highest level of risk of developing severe cell changes and cell abnormalities. This is due to the fact that a negative result for L1 mRNA expression is directly indicative of integrated HPV, and therefore a higher probability of high and constant expression of E6 and E7. Integration of a virus in the human genome has also a direct impact on the stability of the cells. Integration of HPV also reduces the possibility of regression of cell changes.
Women positive for expression of E6 mRNA from one of HPV types 16, 18, 31 or 33 and positive for expression of L1 mRNA have a “high risk” HPV expression and it is still possible that the HPV has been integrated. However, the risk of these women is not classed as high as those who are L1 negative and E6 positive, since there is a reasonable probability that they do not have integrated HPV.
Women negative for expression of E6 mRNA from HPV types 16, 18, 31 or 33 but positive for expression of E6 mRNA from another HPV type, e.g. 35, 39, 45, 52, 56, 58, 59, 66 and 68, are still considered “at risk” and may therefore be placed in risk categories 1 or 2 (as defined above) depending on whether they are positive or negative for expression of L1 mRNA.
Women positive for L1 mRNA but negative for E6 mRNA are scored as being at moderate risk. There may be high-risk HPV types in the sample and L1 expression is indicative of lytic activity. There may also be integrated HPV types but only with viruses that are rare. However, detection of lytic activity may show that the cell may soon develop some changes.
In the wider context of cervical screening the method of the invention may be used to classify women according to risk of developing cervical carcinoma and therefore provide a basis for decisions concerning treatment and/or further screening. By way of example: women in risk category 1, particularly those who exhibit positive expression of E6 mRNA from at least one of HPV types 16, 18, 31 or 33, might be identified as requiring “immediate action”, meaning conisation or colposcopy, including a biopsy and histology.
Women in risk category 2, as defined above, might be scored as requiring immediate attention, meaning colposcopy alone or colposcopy including a biopsy and histology.
Women in risk category 3, as defined above, might be scored as requiring immediate re-test, meaning recall for a further test for HPV expression immediately or after a relatively short interval, e.g. six months.
Women in risk category 4, as defined above, might be returned to the screening program, to be re-tested for HPV expression at a later date.
In a further embodiment the invention provides an in vitro method of screening human subjects for the presence of integrated HPV or a modified episomal HPV genome, which method comprises screening the subject for expression of mRNA transcripts from the L1 gene and the E6 gene of human papillomavirus, wherein subjects negative for expression of L1 mRNA but positive for expression of E6 mRNA are scored as carrying integrated HPV.
The term “integrated HPV” refers to an HPV genome which is integrated into the human genome.
The term “modified episomal HPV genome” is taken to mean an HPV genome which is retained within a cell of the human subject as an episome, i.e. not integrated into the human genome, and which carries a modification as compared to the equivalent wild-type HPV genome, which modification leads to constitutive or persistent expression of transcripts of the E6 and/or E7 genes. The “modification” will typically be a deletion, a multimerisation or concatermerisation of the episome, a re-arrangement of the episome etc affecting the regulation of E6/E7 expression.
As aforesaid, the presence of integrated HPV or a modified episomal HPV genome is indicated by a negative result for L1 mRNA expression, together with a positive result for expression of E6 mRNA in cells of the cervix. Therefore, the ability to predict the presence of integrated HPV or a modified episomal HPV genome in this assay is critically dependent on the ability to score a negative result for L1 mRNA expression. This requires a detection technique which has maximal sensitivity, yet produces minimal false-negative results. In a preferred embodiment this is achieved by using a sensitive amplification and real-time detection technique to screen for the presence or absence of L1 mRNA. The most preferred technique is real-time NASBA amplification using molecular beacons probes, as described by Leone et al., Nucleic Acids Research., 1998, Vol 26, 2150-2155. Due to the sensitivity of this technique the occurrence of false-negative results is minimised and a result of “negative L1 expression” can be scored with greater confidence.
In a further embodiment, a method of screening human subjects for the presence of integrated HPV or a modified episomal HPV genome may be based on screening for expression of E6 mRNA alone. Thus, the invention relates to an in vitro method of screening human subjects for the presence of integrated HPV or a modified episomal HPV genome, which method comprises screening the subject for expression of mRNA transcripts from the E6 gene of human papillomavirus, wherein subjects positive for expression of E6 mRNA are scored as carrying integrated HPV or a modified episomal HPV genome.
Moreover, individuals may be sorted into one of two categories of risk for development of cervical carcinoma based on an “on/off” determination of expression of E6 mRNA alone. Therefore, the invention provides an in vitro method of screening human subjects to assess their risk of developing cervical carcinoma, which method comprises screening the subject for expression of mRNA transcripts of the E6 gene of HPV and sorting the subject into one of two categories of risk for development of cervical carcinoma based on expression of E6 mRNA, wherein individuals positive for expression of E6 mRNA are scored as carrying integrated HPV or a modified episomal HPV genome and are therefore classified as “high risk” for development of cervical carcinoma, whereas individuals negative for expression of E6 mRNA are scored as not carrying integrated HPV or a modified episomal HPV genome and are therefore classified as “no detectable risk” for development of cervical carcinoma.
Subjects are sorted into one of two categories of risk for development of cervical carcinoma based on an “on/off” determination of expression of E6 mRNA in cells of the cervix. Individuals positive for expression of E6 mRNA are scored as carrying integrated HPV or a modified episomal HPV genome and are therefore classified “high risk” for development of cervical carcinoma, whereas individuals negative for expression of E6 mRNA are scored as not carrying integrated HPV a modified episomal HPV genome and are therefore classified as “no detectable risk” for development of cervical carcinoma.
In the context of cervical screening classification of subjects into the two groups having “high risk” or “no detectable risk” for development of cervical carcinoma provides a basis for decisions concerning treatment and/or further screening. For example subjects in the high risk category may be scored as requiring immediate further analysis, e.g. by histological colposcopy, whilst those in the no detectable risk category may be referred back to the screening program at three or five year intervals.
These methods are particularly useful for assessing risk of developing carcinoma in subjects known to be infected with HPV, e.g. those testing positive for HPV DNA, or subjects who have previously manifested a cervical abnormality via cytology or pap smear. Subjects placed in the “no detectable risk” category on the basis of E6 mRNA expression may have HPV DNA present but the negative result for E6 expression indicates that HPV is unrelated to oncogene activity at the time of testing.
The presence of integrated HPV or a modified episomal HPV genome, as indicated by a positive result for E6 mRNA expression, is itself indicative that the subject has abnormal cell changes in the cervix. Therefore, the invention also relates to an in vitro method of identifying human subjects having abnormal cell changes in the cervix, which method comprises screening the subject for expression of mRNA transcripts of the E6 gene of HPV, wherein individuals positive for expression of E6 mRNA are identified as having abnormal cell changes in the cervix.
The term “abnormal cell changes in the cervix” encompasses cell changes which are characteristic of more severe disease than low-grade cervical lesions or low squamous intraepithelial lesions, includes cell changes which are characteristic of disease of equal or greater severity than high-grade CIN (defined as a neoplastic expansion of transformed cells), CIN (cervical intraepithelial neoplasia) III, or high squamous intraepithelial neoplasia (HSIL), including lesions with multiploid DNA profile and “malignant” CIN lesions with increased mean DNA-index values, high percentage of DNA-aneuploidy and 2.5c Exceeding Rates (Hanselaar et al., 1992, Anal Cell Pathol.,
4:315-324; Rihet et al., 1996, J. Clin Pathol 49:892-896; and McDermott et al., 1997, Br. J. Obstet. Gynaecol. 104:623-625).
Cervical Intraepithelial Neoplasia (abbreviated “CIN”), also called Cervical Dysplasia, is a cervical condition caused Human Papilloma Virus. CIN is classified as I, II or III depending on its severity. It is considered a pre-cancerous abnormality, but not an actual cancer. The mildest form, CIN I, usually goes away on its own, although rarely it can progress to cancer. The more severe forms, CIN II and CIN III, most often stay the same or get worse with time. They can become a cancer, but almost never do if treated adequately.
HPV has been identified as a causative agent in development of cellular changes in the cervix, which may lead to the development of cervical carcinoma. These cellular changes are associated with constitutive or persistent expression of E6/E7 proteins from the HPV viral genome. Thus, it is possible to conclude that subjects in which expression of E6 mRNA can be detected, particularly those subjects who exhibit persistent E6 expression when assessed over a period of time, already manifest cellular changes in the cervix. These changes may have taken place in only a very few cells of the cervix, and may not be detectable by conventional cytology. Nevertheless, with the use of sensitive, specific and accurate methods for detection of E6 mRNA it is possible to identify those subjects who already exhibit cellular changes in the cervix at a much earlier stage than would be possible using conventional cytological screening. This will allow earlier intervention with treatments aimed at preventing the development of cervical carcinoma.
As a result of HPV integration into the human genome or as a result of the “modification” in a modified episomal HPV genome, normal control of the viral E6/E7 oncogene transcription is lost (Durst et al., 1985, J Gen Virol, 66(Pt 7): 1515-1522; Pater and Pater, 1985 Virology 145:313-318; Schwarz et al., 1985, Nature 314: 111-114; Park et al., 1997, ibid). In contrast, in premalignant lesions and HPV-infected normal epithelium papillomaviruses predominate in “unmodified” episomal forms, hence oncogene (E6/E7) transcription may be absent or efficiently down-regulated (Johnson et al., 1990, J Gen Virol, 71(Pt 7): 1473-1479; Falcinelli et al., 1993, J Med Virol, 40: 261-265). Integration of human papillomavirus type 16 DNA into the human genome is observed to lead to a more unstable cell activity/genome, and increased stability of E6 and E7 mRNAs (Jeon and Lambert, 1995, Proc Natl Acad Sci USA 92: 1654-1658). Thus HPV integration, typically found in cervical cancers but only infrequently found in CIN lesions (Carmody et al., 1996, Mol Cell Probes, 10: 107-116), appears to be an important event in cervical carcinogenesis.
The present methods detect E6/E7 viral mRNA expression in the cervix instead of DNA. E6/E7 viral expression in cervical cells is a much more accurate assessment of the risk of developing cancer than simply showing that the HPV virus is present. Furthermore, the detection of HPV oncogene transcripts may be a more sensitive indicator of the direct involvement of viral oncogenes in carcinogenesis (Rose et al., 1994, Gynecol Oncol, 52: 212-217; Rose et al., 1995, Gynecol Oncol, 56: 239-244). Detection of E6/E7 transcripts by amplification and detection is a useful diagnostic tool for risk evaluations regarding the development of CIN and its progression to cervical cancer, especially in high-risk HPV type-infected patients with ASCUS and CIN I (Sotlar et al., 1998, Gynecol Oncol, 69: 114-121; Selinka et al., 1998, Lab Invest, 78: 9-18).
The expression of E6/E7 transcripts of HPV-16/18 is uniformly correlated with the physical status of HPV DNAs (Park et al., 1997, Gynecol Oncol, Vol: 65(1), 121-9). In most cervical carcinoma cells the E6 and E7 genes of specific human papillomaviruses are transcribed from viral sequences integrated into host cell chromosomes (von Kleben Doeberitz et al., 1991, Proc Natl Acad Sci USA. Vol: 88(4), 1411-5). Viral load and integration has been evaluated in a large series of CIN lesions (Pietsaro et al., 2002, J Clin Microbiol, Vol: 40(3), 886-91). Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Seventeen of 37 invasive cervical carcinoma samples were identified previously as containing the completely integrated HPV16 genome by using PCR covering the entire E1/E2 gene, and this was confirmed by rliPCR in 16 cases. One case, however, showed a low level of episomal deoxyribonucleic acid in addition to the predominant integrated form. Of the remaining 20 carcinoma samples showing episomal forms in the previous analysis, 14 were found to contain integrated forms using rliPCR, and four contained multimeric (modified) episomal forms. Thus, in total, 31 of 37 of the carcinomas (84%) showed integrated HPV16 genome, while absence of integration could not be detected. (Kalantari et al, 2001, Diagn Mol Pathol, Vol: 10(1), 46-54).
There have been virtually no observations that cervical carcinoma cells exist without integrated HPV or modified episomal HPV DNA (Kalantari et al. 2001; Pietsaro et al., 2002, ibid). It has further been shown that E6 and E7 may only be transcribed from integrated or modified episomal HPV DNA (von Kleben Doeberitz et al., 1991, ibid). Therefore, the inventors surmise that detection of E6/E7 expression provides a direct indication of integrated HPV or modified episomal HPV and high oncogene activity, and conclude that in a clinical context detection of E6 (E6/E7) expression alone is sufficient to identify subjects at “high risk” of developing cervical carcinoma. In other words, if E6/E7 mRNA expression can be detected in a cervical sample, this is directly indicative of cellular abnormalities in the cervix and there is a very high risk of development of cervical carcinoma due to persistent HPV oncogene activity. Therefore, detection of E6/E7 mRNA in a human subject indicates that the subject has a very high risk of developing cervical carcinoma and should undergo immediate further screening, e.g. by colposcopy.
If HPV E6/E7 mRNA expression is not detected, the subject may still have an HPV infection. However due to absence of integration and oncogene activity, it may regress spontaneously (as observed by Pietsaro et al., 2002, ibid).
In a clinical context the performance of methods which rely on screening for expression of E6 mRNA alone is critically dependent on the ability to score a negative result for E6 mRNA expression with confidence. This again requires a detection technique which has maximal sensitivity, yet produces minimal false-negative results. In a preferred embodiment this is achieved by using a sensitive amplification and real-time detection technique to screen for the presence or absence of E6 mRNA. The most preferred technique is real-time NASBA amplification using molecular beacons probes, as described by Leone et al., Nucleic Acids Research., 1998, Vol 26, 2150-2155. Due to the sensitivity of this technique the occurrence of false-negative results is minimised and a result of “negative E6 expression” can be scored with greater confidence. This is extremely important if the assays are to be used in the context of a clinical screening program.
In the methods based on detection of E6 mRNA alone it is preferred to detect at least types HPV 16, 18, 31, 33 and 45, and in a preferred embodiment the assay may detect only these HPV types. DNA from HPV types 16, 18, 31 and 33 has been detected in more than 87% of cervical carcinoma samples (Karlsen et al., 1996, J Clin Microbiol, 34:2095-2100). Other studies have shown that E6 and E7 are almost invariably retained in cervical cancers, as their expression is likely to be necessary for conversion to and maintenance of the malignant state (Choo et al., 1987, J Med Virol 21:101-107; Durst et al., 1995, Cancer Genet Cytogenet, 85: 105-112). In contrast to HPV detection systems which are based on detection of the undamaged genome or the L1 gene sequence, detection of HPV mRNA expressed from the E6/E7 area may detect more than 90% of the patients directly related to a risk of developing cervical carcinoma.
In the clinic, methods based on detection of E6 mRNA are preferred for use in post-screening, i.e. further analysis of individuals having a previous diagnosis of ASCUS, CIN 1 or Condyloma. The method may be used to select those with a high risk of developing cervical carcinoma from amongst the group of individuals having a previous diagnosis of ASCUS, CIN 1 or Condyloma. ASCUS, Condyloma and CIN I may be defined as more or less the same diagnosis due to very low reproducibility between different cytologists and different cytological departments. Östör (Int J. Gyn Path. 12:186-192. 1993) found that only around 1% of the CIN 1 cases may progress to cervical carcinoma. Thus, there is a genuine need for an efficient method of identifying the subset of individuals with ASCUS, Condyloma or CIN I who are at substantial risk of developing cervical carcinoma. One of HPV types 16, 18, 31 or 33 was detected in 87% of the cervical carcinoma cases study by Karlsen et al., 1996. By inclusion of HPV 45, nearly 90% of the cervical carcinoma samples are found to be related to these five HPV types. Therefore, calculated from the data provided by Östör (Int J. Gyn Path. 12:186-192. 1993) more than 99.9% are detected cases with ASCUS, CIN I or condyloma are missed by our HPV-Proofer kit.
In the methods of the invention “positive expression” of an mRNA is taken to mean expression above background. There is no absolute requirement for accurate quantitative determination of the level of mRNA expression or for accurate determination of the relative levels of expression of L1 and E6 mRNA.
In certain embodiments, the methods of the invention may comprise a quantitative determination of levels of mRNA expression. In a preferred embodiment in order to provide a clear distinction between “positive expression” and “negative expression” a determination of “positive expression” may require the presence of more than 50 copies of the relevant mRNA (per ml of sample or per total volume of sample), whereas a determination of “negative expression” may require the presence of less than 1 copy of the relevant mRNA (per ml of sample or per total volume of sample).
The methods of the invention will preferably involve screening for E6 mRNA using a technique which is able to detect specifically E6 mRNA from cancer-associated HPV types, more preferably “high risk” cancer-associated HPV types. In the most preferred embodiment the methods involve screening for E6 mRNA using a technique which is able to detect E6 mRNA from HPV types 16, 18, 31 and 33, and preferably also 45. Most preferably, the method will specifically detect expression of E6 mRNA from at least one of HPV types 16, 18, 31, 33, and preferably also 45, and most preferably all five types. However, women positive for positive for expression of E6 from other types than 16, 18, 31, 33 and 45, e.g. 35, 39, 45, 52, 56, 58, 59, 66 and 68 may still be “at risk” of developing cervical carcinoma. Thus, the method may encompass screening for expression of E6 mRNA from one or more of these HPV types, most preferably in addition to screening for E6 mRNA from HPV types 16, 18, 31, 33 and 45. Certain HPV types exhibit a marked geographical/population distribution. Therefore, it may be appropriate to include primers specific for an HPV type known to be prevalent in the population/geographical area under test, for example in addition to screening for HPV types 16, 18, 31, 33 and 45.
For the avoidance of doubt, unless otherwise stated the term “E6 mRNA” as used herein encompasses all naturally occurring mRNA transcripts which contain all or part of the E6 open reading frame, including naturally occurring splice variants, and therefore includes transcripts which additionally contain all or part of the E7 open reading frame (and indeed further open reading frames). The terms “E6/E7 mRNA”, “E6/E7 transcripts” etc are used interchangeably with the terms “E6 mRNA”, “E6 transcripts” and also encompass naturally occurring mRNA transcripts which contain all or part of the E6 open reading frame, including naturally occurring splice variants, and transcripts which contain all or part of the E7 open reading frame. The term “oncogene expression”, unless otherwise stated, also refers to naturally occurring mRNA transcripts which contain all or part of the E6 open reading frame, including naturally occurring splice variants, and transcripts which contain all or part of the E7 open reading frame.
Four E6/E7 mRNA species have so far been described in cells infected with HPV 16, namely an unspliced E6 transcript and three spliced transcripts denoted E6*I, E6*II and E6*III (Smotkin D, et al., J. Virol. 1989 March 63(3):1441-7; Smotkin D, Wettstein F O. Proc Natl Acad Sci USA. 1986 July 83(13):4680-4; Doorbar J. et al., Virology. 1990 September 178(1):254-62; Cornelissen M T, et al. J Gen Virol. 1990 May 71(Pt 5):1243-6; Johnson M A, et al. J Gen Virol. 1990 July 71(Pt 7):1473-9; Schneider-Maunoury S, et al. J. Virol. 1987 October 61(10):3295-8; Sherman L, et al. Int J. Cancer. 1992 February 50(3):356-64). All four transcripts are transcribed from a single promoter (p97) located just upstream of the second ATG of the E6 ORF.
In one embodiment the methods may comprise screening for E6 transcripts which contain all or part of the E7 open reading frame, This may be accomplished, for example, using primers or probes specific for the E7 coding region.
In a further embodiment, the methods may comprise screening for the presence of “full length” E6 transcripts. In the case of HPV 16 the term “full length E6 transcripts” refers to transcripts which contain all of the region from nucleotide (nt) 97 to nt 880 in the E6 ORF, inclusive of nt 97 and 880. Nucleotide positions are numbered according to standard HPV nomenclature (see Human Papillomavirus Compendium OnLine, available via the internet or in paper form from HV Database, Mail Stop K710, Los Alamos National Laboratory, Los Alamos, N. Mex. 87545, USA). Specific detection of full length transcripts may be accomplished, for example, using primers or probes which are specific for the region which is present only in full length E6 transcripts, not in splice variants. Different HPV types exhibit different patterns of E6/E7 mRNA expression. Transcript maps for various HPV types, including HPV types 16 and 31, which may be used to assist in the design of probes or primers for detection of E6/E7 transcripts are publicly available via the Human Papillomavirus Compendium (as above).
E6 oligonucleotide primers are described herein which are suitable for use in amplification of regions of the E6 mRNA from various HPV types by NASBA or PCR.
In a preferred embodiment methods which involve screening for L1 mRNA expression may comprise screening for L1 mRNA expression using a technique which is able to detect L1 mRNA from substantially all known HPV types or at least the major cancer-associated HPV types (e.g. preferably all of HPV types 16, 18, 31 and 33). L1 primers and probes are described herein which are capable of detecting L1 mRNA from HPV types 6, 11, 16, 18, 31, 33, 35 and 51 in cervical samples.
Detection of L1 transcripts can be said to detect HPV “virulence”, meaning the presence of HPV lytic activity. Detection of E6/E7 transcripts can be said to detect HPV “pathogenesis” since expression of these mRNAs is indicative of molecular events associated with risk of developing carcinoma.
In a study of 4589 women it was possible to detect all except one case of CIN III lesions or cancer using a method based on screening for expression of E6 and L1 mRNA (see accompanying Examples).
In further embodiments, the above-described methods of the invention may comprise screening for expression of mRNA transcripts from the human p16ink4a gene, in addition to screening for expression of HPV L1 and/or E6 transcripts.
A positive result for expression of p16ink4a mRNA is taken as a further indication of risk of developing cervical carcinoma.
P16ink4a, and the related family members, may function to regulate the phosphorylation and the growth suppressive activity of the restinoblastoma gene product (RB). In support of this, it has been found that there is an inverse relationship between the expression of p16ink4a protein and the presence of normal RB in selected cancer cell lines; p16ink4a protein is detectable when RB is mutant, deleted, or inactivated, and it is markedly reduced or absent in cell lines that contain a normal RB. Kheif et al. (Kheif S N et al., Proc. Natl. Acad. Sci. USA 93:4350-4354. 1996), found that p16ink4a protein is expressed in human cervical carcinoma cells that contain either a mutant RB or a wild-type RB that is functionally inactivated by E7. They also show that the inactivation of RB correlates with an upregulation of p16ink4a confirming a feedback loop involving p16ink4a and RB. Milde-Langosch et al. (Milde-Langosch K, et al., (2001) Virchows Arch 439: 55-61) found that there were significant correlations between strong p16 expression and HPV16/18 infection and between strong p16 expression and HPV 16/18 E6/E7 oncogene expression. Klaes et al., (Klaes R, et al., (2001) Int J Cancer 92: 276-284) observed a strong over expression of the p16ink4a gene product in 150 of 152 high-grade dysplastic cervical lesions (CIN II to invasive cancer), whereas normal cervical epithelium or inflammatory or metaplastic lesions were not stained with the p16ink4a specific monoclonal antibody E6H4. All CIN I scored lesions associated with LR-HPV types displayed no or only focal or sporadic reactivity, whereas all but two CIN I scored lesions associated with HR-HPV types showed strong and diffuse staining for p16ink4a.
The disclosed screening methods may be carried out on a preparation of nucleic acid isolated from a clinical sample or biopsy containing cervical cells taken from the subject under test. Suitable samples which may be used as a source of nucleic acid include (but not exclusively) cervical swabs, cervical biopsies, cervical scrapings, skin biopsies/warts, also paraffin embedded tissues, and formalin or methanol fixed cells.
The preparation of nucleic acid to be screened using the disclosed method must include mRNA, however it need not be a preparation of purified poly A+ mRNA and preparations of total RNA or crude preparations of total nucleic acid containing both RNA and genomic DNA, or even crude cell lysates are also suitable as starting material for a NASBA reaction. Essentially any technique known in the art for the isolation of a preparation of nucleic acid including mRNA may be used to isolate nucleic acid from a test sample. A preferred technique is the “Boom” isolation method described in U.S. Pat. No. 5,234,809 and EP-B-0389,063. This method, which can be used to isolate a nucleic acid preparation containing both RNA and DNA, is based on the nucleic acid binding properties of silicon dioxide particles in the presence of the chaotropic agent guanidine thiocyanate (GuSCN).
The methods of the invention are based on assessment of active transcription of the HPV genome in cervical cells. The methods are not limited with respect to the precise technique used to detect mRNA expression. Many techniques for detection of specific mRNA sequences are known in the art and may be used in accordance with the invention. For example, specific mRNAs may be detected by hybridisation, amplification or sequencing techniques.
It is most preferred to detect mRNA expression by means of an amplification technique, most preferably an isothermal amplification such as NASBA, transcription-mediated amplification, signal-mediated amplification of RNA technology, isothermal solution phase amplification, etc. All of these methods are well known in the art More preferably mRNA expression is detected by an isothermal amplification in combination with real-time detection of the amplification product. The most preferred combination is amplification by NASBA, coupled with real-time detection of the amplification product using molecular beacons technology, as described by Leone et al., Nucleic Acids Research, 1998, Vol 26, 2150-2155.
Methods for the detection of HPV in a test sample using the NASBA technique will generally comprise the following steps:
(a) assembling a reaction medium comprising suitable primer-pairs, an RNA directed DNA polymerase, a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA, an RNA polymerase that recognises said promoter, and ribonucleoside and deoxyribonucleoside triphosphates;
(b) incubating the reaction medium with a preparation of nucleic acid isolated from a test sample suspected of containing HPV under reaction conditions which permit a NASBA amplification reaction; and
(c) detecting and/or quantitatively measuring any HPV-specific product of the NASBA amplification reaction.
Detection of the specific product(s) of the NASBA reaction (i.e. sense and/or antisense copies of the target RNA) may be carried out in a number of different ways. In one approach the NASBA product(s) may be detected with the use of an HPV-specific hybridisation probe capable of specifically annealing to the NASBA product. The hybridisation probe may be attached to a revealing label, for example a fluorescent, luminescent, radioactive or chemiluminescent compound or an enzyme label or any other type of label known to those of ordinary skill in the art. The precise nature of the label is not critical, but it should be capable of producing a signal detectable by external means, either by itself or in conjunction with one or more additional substances (e.g. the substrate for an enzyme).
A preferred detection method is so-called “real-time NASBA” which allows continuous monitoring of the formation of the product of the NASBA reaction over the course of the reaction. In a preferred embodiment this may be achieved using a “molecular beacons” probe comprising an HPV-specific sequence capable of annealing to the NASBA product, a stem-duplex forming oligonucleotide sequence and a pair of fluorescer/quencher moieties, as known in the art and described herein. If the molecular beacons probe is added to the reaction mixture prior to amplification it may be possible to monitor the formation of the NASBA product in real-time (Leone et al., Nucleic Acids Research, 1998, Vol 26, 2150-2155). Reagent kits and instrumentation for performing real-time NASBA detection are available commercially (e.g. NucliSens™ EasyQ system, from Organon Teknika).
In a further approach, the molecular beacons technology may be incorporated into the primer 2 oligonucleotide allowing real-time monitoring of the NASBA reaction without the need for a separate hybridisation probe.
In a still further approach the products of the NASBA reaction may be monitored using a generic labelled detection probe which hybridises to a nucleotide sequence in the 5′ terminus of the primer 2 oligonucleotide. This is equivalent to the “NucliSens™” detection system supplied by Organon Teknika. In this system specificity for NASBA products derived from the target HPV mRNA may be conferred by using HPV-specific capture probes comprising probe oligonucleotides as described herein attached to a solid support such as a magnetic microbead. Most preferably the generic labelled detection probe is the ECL™ detection probe supplied by Organon Teknika. NASBA amplicons are hybridized to the HPV-specific capture probes and the generic ECL probe (via a complementary sequence on primer 2). Following hybridization the bead/amplicon/ECL probe complexes may be captured at the magnet electrode of an automatic ECL reader (e.g. the NucliSens™ reader supplied by Organon Teknika). Subsequently, a voltage pulse triggers the ECL™ reaction.
The detection of HPV mRNA is also of clinical relevance in cancers other than cervical carcinoma including, for example, head and neck carcinoma, oral and tongue carcinoma, skin carcinoma, anal and vaginal carcinoma. Detection of HPV mRNA may also be very useful in the diagnosis of micrometastases in lymph nodes in the lower part of the body. Hence, the invention also contemplates screens for susceptibility to the above-listed cancers based on screening for expression of HPV L1 and E6 transcripts.
In accordance with a further aspect of the invention there is provided a kit for use in the detection of transcripts of the L1 and E6 genes of HPV, the kit comprising at least one primer-pair suitable for use in amplification of a region of L1 transcripts from at least HPV types 16, 18, 31 and 33, and preferably also HPV 45, and one or more primer-pairs which enable amplification of a region of E6 transcripts from HPV types 16, 18, 31 and 33, and preferably also HPV 45.
“Primer-pair” taken to mean are pair of primers which may be used in combination to amplify a specific region of the L1 or E6 mRNA using any known nucleic acid technique. In preferred embodiments the primer-pairs included in the kit will be suitable for use in NASBA amplification or similar isothermal amplification techniques.
The individual primers making up each primer-pair included in the kit may be supplied separately (e.g. a separate container of each primer) or, more preferably, may be supplied mixed in a single container. Combinations of two or more primer-pairs may be supplied ready-mixed in a single container within the kit. It may be convenient to supply two or more primer-pairs in a single container where the two or more amplification reactions are to be “multiplexed”, meaning performed simultaneously in a single reaction vessel.
The primer-pair(s) suitable for use in amplification of a region of E6 transcripts should enable amplification a region of E6 mRNA from at least the major cancer-associated HPV types 16, 18, 31 and 33, and preferably also HPV 45. There are several different ways in which this can be achieved.
In one embodiment, the kit may contain separate primer-pairs specific for each of HPV types 16, 18, 31 and 33, and preferably also HPV 45. These primer-pairs may be supplied within the kit in separate containers, or they may be supplied as mixtures of two or more primer-pairs in a single container, for example to enable multiplexing of the amplification reactions.
In a further embodiment, the kit may contain a single primer-pair capable of amplifying a region of the E6 gene from HPV types 16, 18, 31 and 33, and preferably also HPV 45, which thus enables amplification of all four (preferably five) types in a single amplification reaction. This could, for example, be achieved with the use of a pair of degenerate primers or by selection of a region of the E6 mRNA which is highly conserved across HPV types.
The E6 primer-pair may correspond to any region of the E6 mRNA, an may enable amplification of all or part of the E6 open reading frame and/or the E7 open reading frame.
The kit may further include primer-pairs suitable for use in amplification of E6 mRNA from HPV types other than types 16, 18, 31 and 33, and preferably also HPV 45. For example, the kit may be supplemented with E6 primers for detection of an HPV type which is endemic in a particular geographical area or population.
The primer-pair(s) suitable for use in amplification of a region of L1 transcripts should be capable of amplifying a region of L1 mRNA from at least the major cancer-associated HPV types 16, 18, 31 and 33, and preferably also HPV 45, and will preferably be suitable for use in amplification of a region of L1 mRNAs from substantially all known HPV types. With the use of such primers it is possible to test for active transcription of L1 mRNA from multiple HPV types in a single amplification reaction.
It is possible to design primers capable of detecting L1 transcripts from multiple HPV types by selecting regions of the L1 transcript which are highly conserved.
In a further approach, specificity for multiple HPV types may be achieved with the use of degenerate oligonucleotide primers or complex mixtures of polynucleotides which exhibit minor sequence variations, preferably corresponding to sites of sequence variation between HPV genotypes. The rationale behind the use of such degenerate primers or mixtures is that the mixture may contain at least one primer-pair capable of detecting each HPV type.
In a still further approach specificity for multiple HPV types may be achieved by incorporating into the primers one or more inosine nucleotides, preferably at sites of sequence variation between HPV genotypes.
The E6 and L1 primer-pairs may be supplied in separate containers within the kit, or the L1 primer-pair(s) may be supplied as a mixture with one or more E6 primer-pairs in a single container.
The kits may further comprise one or more probes suitable for use in detection of the products of amplification reactions carried out using the primer-pairs included within the kit. The probe(s) may be supplied as a separate reagent within the kit. Alternatively, the probe(s) may be supplied as a mixture with one or more primer-pairs.
The primers and probes included in the kit are preferably single stranded DNA molecules. Non-natural synthetic polynucleotides which retain the ability to base-pair with a complementary nucleic acid molecule may also be used, including synthetic oligonucleotides which incorporate modified bases and synthetic oligonucleotides wherein the links between individual nucleosides include bonds other than phosphodiester bonds. The primers and probes may be produced according to techniques well known in the art, such as by chemical synthesis using standard apparatus and protocols for oligonucleotide synthesis.
The primers and probes will typically be isolated single-stranded polynucleotides of no more than 100 bases in length, more typically less than 55 bases in length. For the avoidance of doubt it is hereby stated that the terms “primer” and “probe” exclude naturally occurring full-length HPV genomes.
Several general types of oligonucleotide primers and probes incorporating HPV-specific sequences may be included in the kit. Typically, such primers and probes may comprise additional, non-HPV sequences, for example sequences which are required for an amplification reaction or which facilitate detection of the products of the amplification reaction.
The first type of primers are primer 1 oligonucleotides (also referred to herein as NASBA P1 primers), which are oligonucleotides of generally approximately 50 bases in length, containing an average of about 20 bases at the 3′ end that are complementary to a region of the target mRNA. Oligonucleotides suitable for use as NASBA P1 primers are denoted “P1/PCR” in Table 1. P1 primer oligonucleotides have the general structure X1-SEQ, wherein SEQ represents an HPV-specific sequence and X1 is a sequence comprising a promoter that is recognized by a specific RNA polymerase. Bacteriophage promoters, for example the T7, T3 and SP6 promoters, are preferred for use in the oligonucleotides of the invention, since they provide advantages of high level transcription which is dependent only on binding of the appropriate RNA polymerase. In a preferred embodiment, sequence “X1” may comprise the sequence AATTCTAATACGACTCACTATAGGG (SEQ ID No 171) or the sequence AATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID No 172). These sequences contains a T7 promoter, including the transcription initiation site for T7 RNA polymerase.
The HPV-specific sequences in the primers denoted in Table 1 as “P1/PCR” may also be adapted for use in standard PCR primers. When these sequences are used as the basis of NASBA P1 primers they have the general structure X1-SEQ, as defined above. The promoter sequence X1 is essential in a NASBA P1 primer. However, when the same sequences are used as the basis of standard PCR primers it is not necessary to include X1.
A second type of primers are NASBA primer 2 oligonucleotides (also referred to herein as NASBA P2 primers) which generally comprise a sequence of approximately 20 bases substantially identical to a region of the target mRNA. The oligonucleotide sequences denoted in Table 1 as “P2/PCR” are suitable for use in both NASBA P2 primers and standard PCR primers.
Oligonucleotides intended for use as NASBA P2 primers may, in a particular but non-limiting embodiment, further comprise a sequence of nucleotides at the 5′ end which is unrelated to the target mRNA but which is capable of hybridising to a generic detection probe. The detection probe will preferably be labelled, for example with a fluorescent, luminescent or enzymatic label. In one embodiment the detection probe is labelled with a label that permits detection using ECL™ technology, although it will be appreciated that the invention is in no way limited to this particular method of detection. In a preferred embodiment the 5′ end of the primer 2 oligonucleotides may comprise the sequence GATGCAAGGTCGCATATGAG (SEQ ID No 170). This sequence is capable of hybridising to a generic ECL™ probe commercially available from Organon Teknika having the following structure:
Ru(bpy)32+-GAT GCA AGG TCG CAT ATG AG-3′
In a different embodiment the primer 2 oligonucleotide may incorporate “molecular beacons” technology, which is known in the art and described, for example, in WO 95/13399 by Tyagi and Kramer, Nature Biotechnology. 14: 303-308, 1996, to allow for real-time monitoring of the NASBA reaction.
Target-specific probe oligonucleotides may also be included within the kit. Probe oligonucleotides generally comprise a sequence of approximately 20-25 bases substantially identical to a region of the target mRNA, or the complement thereof. Example HPV-specific oligonucleotide sequences which are suitable for use as probes are denoted “PO” in Table 1. The probe oligonucleotides may be used as target-specific hybridisation probes for detection of the products of a NASBA or PCR reaction. In this connection the probe oligonucleotides may be coupled to a solid support, such as paramagnetic beads, to form a capture probe (see below). In a preferred embodiment the 5′ end of the probe oligonucleotide may be labelled with biotin. The addition of a biotin label facilitates attachment of the probe to a solid support via a biotin/streptavidin or biotin/avidin linkage.
Target-specific probes enabling real-time detection of amplification products may incorporate “molecular beacons” technology which is known in the art and described, for example, by Tyagi and Kramer, Nature Biotechnology. 14: 303-308, 1996 and in WO 95/13399. Example HPV-specific oligonucleotide sequences suitable for use as molecular beacons probes are denoted “MB” in Table 1.
The term “molecular beacons probes” as used herein is taken to mean molecules having the structure:X2-arm1-target-arm2-X3 wherein “target” represents a target-specific sequence of nucleotides, “X2” and “X3” represent a fluorescent moiety and a quencher moiety capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two are held together in close proximity and “arm1” and “arm2” represent complementary sequences capable of forming a stem duplex.
Preferred combinations of “arm1” and “arm2” sequences are as follows, however these are intended to be illustrative rather than limiting to the invention:
cgcatg-SEQ-catgcg ccagct-SEQ-agctgg cacgc-SEQ-gcgtg cgatcg-SEQ-cgatcg ccgtcg-SEQ-cgacgg cggacc-SEQ-ggtccg ccgaagg-SEQ-ccttcgg cacgtcg-SEQ-cgacgtg cgcagc-SEQ-gctgcg ccaagc-SEQ-gcttgg ccaagcg-SEQ-cgcttgg cccagc-SEQ-gctggg ccaaagc-SEQ-gctttgg cctgc-SEQ-gcagg ccaccc-SEQ-gggtgg ccaagcc-SEQ-ggcttgg ccagcg-SEQ-cgctgg cgcatg-SEQ-catgcg
The use of molecular beacons technology allows for real-time monitoring of amplification reactions, for example NASBA amplification (see Leone et al., Nucleic Acids Research., 1998, vol: 26, pp 2150-2155). The molecular beacons probes generally include complementary sequences flanking the HPV-specific sequence, represented herein by the notation arm1 and arm2, which are capable of hybridising to each other form a stem duplex structure. The precise sequences of arm1 and arm2 are not material to the invention, except for the requirement that these sequences must be capable of forming a stem duplex when the probe is not bound to a target HPV sequence.
Molecular beacons probes also include a fluorescent moiety and a quencher moiety, the fluorescent and the quencher moieties being represented herein by the notation X2 and X3. As will be appreciated be the skilled reader, the fluorescer and quencher moieties are selected such that the quencher moiety is capable of substantially or completely quenching the fluorescence from the fluorescent moiety when the two moieties are in close proximity, e.g. when the probe is in the hairpin “closed” conformation in the absence of the target sequence. Upon binding to the target sequence, the fluorescent and quencher moieties are held apart such that the fluorescence of the fluorescent moiety is no longer quenched.
Many examples of suitable pairs of quencher/fluorescer moieties which may be used in accordance with the invention are known in the art (see WO 95/13399, Tyagi and Kramer, ibid). A broad range of fluorophores in many different colours made be used, including for example 5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), fluorescein, FAM and Texas Red (see Tyagi, Bratu and Kramer, 1998, Nature Biotechnology, 16, 49-53. The use of probes labelled with different coloured fluorophores enables “multiplex” detection of two or more different probes in a single reaction vessel. A preferred quencher is 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), a non-fluorescent chromophore, which serves as a “universal” quencher for a wide range of fluorophores. The fluorescer and quencher moieties may be covalently attached to the probe in either orientation, either with the fluorescer at or near the 5′ end and the quencher at or near the 3′ end or vice versa. Protocols for the synthesis of molecular beacon probes are known in the art. A detailed protocol for synthesis is provided in a paper entitled “Molecular Beacons: Hybridization Probes for Detection of Nucleic Acids in Homogenous Solutions” by Sanjay Tyagi et al., Department of Molecular Genetics, Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA, which is available online via the PHRI website (at phri.nyu.edu or molecular-beacons.org)
Suitable combinations of the NASBA P1 and NASBA P2 primers may be used to drive a NASBA amplification reaction. In order to drive a NASBA amplification reaction the primer 1 and primer 2 oligonucleotides must be capable of priming synthesis of a double-stranded DNA from a target region of mRNA. For this to occur the primer 1 and primer 2 oligonucleotides must comprise target-specific sequences which are complementary to regions of the sense and the antisense strand of the target mRNA, respectively.
In the first phase of the NASBA amplification cycle, the so-called “non-cyclic” phase, the primer 1 oligonucleotide anneals to a complementary sequence in the target mRNA and its 3′ end is extended by the action of an RNA-dependent DNA polymerase (e.g. reverse transcriptase) to form a first-strand cDNA synthesis. The RNA strand of the resulting RNA:DNA hybrid is then digested, e.g. by the action of RNaseH, to leave a single stranded DNA. The primer 2 oligonucleotide anneals to a complementary sequence towards the 3′ end of this single stranded DNA and its 3′ end is extended (by the action of reverse transcriptase), forming a double stranded DNA. RNA polymerase is then able to transcribe multiple RNA copies from the now transcriptionally active promoter sequence within the double-stranded DNA. This RNA transcript, which is antisense to the original target mRNA, can act as a template for a further round of NASBA reactions, with primer 2 annealing to the RNA and priming synthesis of the first cDNA strand and primer 1 priming synthesis of the second cDNA strand. The general principles of the NASBA reaction are well known in the art (see Compton, J. Nature. 350: 91-92).
The target-specific probe oligonucleotides described herein may also be attached to a solid support, such as magnetic microbeads, and used as “capture probes” to immobilise the product of the NASBA amplification reaction (a single stranded RNA). The target-specific “molecular beacons” probes described herein may be used for real-time monitoring of the NASBA reaction.
Kits according to the invention may also including a positive control containing E6 and/or L1 mRNA from a known HPV type. Suitable controls include, for example, nucleic acid extracts prepared from cell lines infected with known HPV types (e.g. HeLa, CaSki).
Kits further may contain internal control amplification primers, e.g. primers specific for human U1A RNA.
Kits containing primers (and optionally probes) suitable for use in NASBA amplification may further comprise a mixture of enzymes required for the NASBA reaction, e.g. enzyme mixture containing an RNA directed DNA polymerase (e.g. a reverse transcriptase), a ribonuclease that hydrolyses the RNA strand of an RNA-DNA hybrid without hydrolysing single or double stranded RNA or DNA (e.g. RNaseH) and an RNA polymerase. The RNA polymerase should be one which recognises the promoter sequence present in the 5′ terminal region of the NASBA P1 primers supplied in the reagent kit. The kit may also comprise a supply of NASBA buffer containing the ribonucleosides and deoxyribonucleosides required for RNA and DNA synthesis. The composition of a standard NASBA reaction buffer will be well known to those skilled in the art (see also Leone et al., ibid).
TABLE 1E6-specific sequences for inclusionin NASBA/PCR primers and probesPrimer/probeHPVSEQ IDtypeSequenceTypent1P2/PCRCCACAGGAGCGACCCAGAAAGTTA16116 2P1/PCRX1-ACGGTTTGTTGTATTGCTGTTC16368 3P2/PCRCCACAGGAGCGACCCAGAAA16116 4P1/PCRX1-GGTTTGTTGTATTGCTGTTC16368 5P1/PCRX1-ATTCCCATCTCTATATACTA16258 6P1/PCRX1-TCACGTCGCAGTAACTGT16208 7P1/PCRX1-TTGCTTGCAGTACACACA16191 8P1/PCRX1-TGCAGTACACACATTCTA16186 9P1/PCRX1-GCAGTACACACATTCTAA16185 10P2/PCRACAGTTATGCACAGAGCT16142 11P2/PCRATATTAGAATGTGTGTAC16182 12P2/PCRTTAGAATGTGTGTACTGC16185 13P2/PCRGAATGTGTGTACTGCAAG16188 14POACAGTTATGCACAGAGCT16142 15POATATTAGAATGTGTGTAC16182 16POTTAGAATGTGTGTACTGC16185 17POGAATGTGTGTACTGCAAG16188 18POCTTTGCTTTTCGGGATTTATGC16235 19POTATGACTTTGCTTTTCGGGA16230 20MBX2-arm1-TATGACTTTGCTTTTCGGGA-arm2-X316230 21P2/PCRCAGAGGAGGAGGATGAAATAGTA16656 22P1/PCRX1-GCACAACCGAAGCGTAGAGTCACAC16741 23POTGGACAAGCAGAACCGGACAGAGC16687 24P2/PCRCAGAGGAGGAGGATGAAATAGA16656 25P1/PCRX1-GCACAACCGAAGCGTAGAGTCA16741 26POAGCAGAACCGGACAGAGCCCATTA16693 27P2/PCRACGATGAAATAGATGGAGTT18702 28P1/PCRX1-CACGGACACACAAAGGACAG18869 28POAGCCGAACCACAACGTCACA18748 30P2/PCRGAAAACGATGAAATAGATGGAG18698 31P1/PCRX1-ACACCACGGACACACAAAGGACAG18869 32POGAACCACAACGTCACACAATG18752 33MBX2-arm1-GAACCACAACGTCACACAATG-18752arm2-X3 34P2/PCRTTCCGGTTGACCTTCTATGT18651 35P1/PCRX1-GGTCGTCTGCTGAGCTTTCT18817 36P2/PCRGCAAGACATAGAAATAACCTG18179 37P1/PCRX1-ACCCAGTGTTAGTTAGTT18379 38POTGCAAGACAGTATTGGAACT18207 39P2/PCRGGAAATACCCTACGATGAAC31164 40P1/PCRX1-GGACACAACGGTCTTTGACA31423 41POATAGGGACGACACACCACACGGAG31268 42P2/PCRGGAAATACCCTACGATGAACTA31164 43P1/PCRX1-CTGGACACAACGGTCTTTGACA31423 44POTAGGGACGACACACCACACGGA31269 45P2/PCRACTGACCTCCACTGTTATGA31617 46P1/PCRX1-TATCTACTTGTGTGCTCTGT31766 47POGACAAGCAGAACCGGACACATC31687 48P2/PCRTGACCTCCACTGTTATGAGCAATT31619 49P1/PCRX1-TGCGAATATCTACTTGTGTGCTCT GT31766 50POGGACAAGCAGAACCGGACACATCCAA31686 51MBX2-arm1-GGACAAGCAGAACCGGACACATCCAA-31686arm2-X3 52P2/PCRACTGACCTCCACTGTTAT31617 53P1/PCRX1-CACGATTCCAAATGAGCCCAT31809 54P2/PCRTATCCTGAACCAACTGACCTAT33618 55P1/PCRX1-TTGACACATAAACGAACTG33763 56POCAGATGGACAAGCACAACC33694 57P2/PCRTCCTGAACCAACTGACCTAT33620 58P1/PCRX1-CCCATAAGTAGTTGCTGTAT33807 59POGGACAAGCACAACCAGCCACAGC33699 60MBX2-arm1-GGACAAGCACAACCAGCCACAGC-33699arm2-X3 61P2/PCRGACCTTTGTGTCCTCAAGAA33431 62P1/PCRX1-AGGTCAGTTGGTTCAGGATA33618 63POAGAAACTGCACTGTGACGTGT33543 64P2/PCRATTACAGCGGAGTGAGGTAT35217 65P1/PCRX1-GTCTTTGCTTTTCAACTGGA35442 66POATAGAGAAGGCCAGCCATAT35270 67P2/PCRTCAGAGGAGGAGGAAGATACTA35655 68P1/PCRX1-GATTATGCTCTCTGTGAACA35844 69P2/PCRCCCGAGGCAACTGACCTATA35610 70P1/PCRX1-GTCAATGTGTGTGCTCTGTA35770 71POGACAAGCAAAACCAGACACCTCCAA35692 72POGACAAGCAAAACCAGACACC35692 73P2/PCRTTGTGTGAGGTGCTGGAAGAAT52144 74P1/PCRX1-CCCTCTCTTCTAATGTTT52358 75POGTGCCTACGCTTTTTATCTA52296 76P2/PCRGTGCCTACGCTTTTTATCTA52296 77P1/PCRX1-GGGGTCTCCAACACTCTGAACA52507 78POTGCAAACAAGCGATTTCA52461 79P2/PCRTCAGGCGTTGGAGACATC58157 80P1/PCRX1-AGCAATCGTAAGCACACT58301 81P2/PCRTCTGTGCATGAAATCGAA58173 82P1/PCRX1-AGCACACTTTACATACTG58291 83POTGAAATGCGTTGAATGCA58192 84POTTGCAGCGATCTGAGGTATATG58218 85P2/PCRTACACTGCTGGACAACATB (11)514 86P1/PCRX1-TCATCTTCTGAGCTGTCTB (11)619 87P2/PCRTACACTGCTGGACAACATGCAB (11)514 88P1/PCRX1-GTCACATCCACAGCAACAGGTCAB (11)693 89POGTAGGGTTACATTGCTATGAB (11)590 90POGTAGGGTTACATTGCTATGAGCB (11)590 91P2/PCRTGACCTGTTGCTGTGGATGTGAB (11)693 92P1/PCRX1-TACCTGAATCGTCCGCCATB (11)832 93POATWGTGTGTCCCATCTGCB (11)794 94P2/PCRCATGCCATAAATGTATAGAC (1829539 45) 95P1/PCRX1-CACCGCAGGCACCTTATTAAC (1840839 45 96POAGAATTAGAGAATTAAGAC (1832439 45 97P2/PCRGCAGACGACCACTACAGCAAA39210 98P1/PCRX1-ACACCGAGTCCGAGTAATA39344 99POATAGGGACGGGGAACCACT39273 100P2/PCRTATTACTCGGACTCGGTGT39344 101P1/PCRX1-CTTGGGTTTCTCTTCGTGTTA39558 102POGGACCACAAAACGGGAGGAC39531 103P2/PCRGAAATAGATGAACCCGACCA39703 104P1/PCRX1-GCACACCACGGACACACAAA39886 105POTAGCCAGACGGGATGAACCACAGC39749 106P2/PCRAACCATTGAACCCAGCAGAAA45430 107P1/PCRX1-TCTTTCTTGCCGTGCCTGGTCA45527 108POGTACCGAGGGCAGTGTAATA45500 109P2/PCRAACCATTGAACCCAGCAGAAA45430 110P1/PCRX1-TCTTTCTTGCCGTGCCTGGTCA45527 111P2/PCRGAAACCATTGAACCCAGCAGAAAA45428 112P1/PCRX1-TTGCTATACTTGTGTTTCCCTACG45558 113POGTACCGAGGGCAGTGTAATA45500 114POGGACAAACGAAGATTTCACA45467 115P2/PCRGTTGACCTGTTGTGTTACCAGCAAT45656 116P1/PCRX1-CACCACGGACACACAAAGGACAAG45868 117P2/PCRCTGTTGACCTGTTGTGTTACGA45654 118P1/PCRX1-CCACGGACACACAAAGGACAAG45868 119P2/PCRGTTGACCTGTTGTGTTACGA45656 120P1/PCRX1-ACGGACACACAAAGGACAAG45868 121POGAGTCAGAGGAGGAAAACGATG45686 122POAGGAAAACGATGAAGCAGATGGAGT45696 123POACAACTACCAGCCCGACGAGCCGAA45730 124P2/PCRGGAGGAGGATGAAGTAGATA51658 125P1/PCRX1-GCCCATTAACATCTGCTGTA51807 126P2/PCRAGAGGAGGAGGATGAAGTAGATA51655 127P1/PCRX1-ACGGGCAAACCAGGCTTAGT51829 128POGCAGGTGTTCAAGTGTAGTA51747 129POTGGCAGTGGAAAGCAGTGGAGACA51771 130P2/PCRTTGGGGTGCTGGAGACAAACATCT56519 131P1/PCRX1-TTCATCCTCATCCTCATCCTCTGA56665 132P2/PCRTGGGGTGCTGGAGACAAACATC56520 133P1/PCRX1-CATCCTCATCCTCATCCTCTGA56665 134P2/PCRTTGGGGTGCTGGAGACAAACAT56519 135P1/PCRX1-CCACAAACTTACACTCACAACA56764 136POAAAGTACCAACGCTGCAAGACGT56581 137POAGAACTAACACCTCAAACAGAAAT56610 138POAGTACCAACGCTGCAAGACGTT56583 139P1/PCRX1-TTGGACAGCTCAGAGGATGAGG56656 140P2/PCRGATTTTCCTTATGCAGTGTG56279 141P1/PCRX1-GACATCTGTAGCACCTTATT56410 142POGACTATTCAGTGTATGGAGC56348 143POCAACTGAYCTMYACTGTTATGAA (1631 35) 144MBX2-arm1-CAACTGAYCTMYACTGTTATGA-A (16arm2-X331 35) 145POGAAMCAACTGACCTAYWCTGCTATA (3352 58) 146MBX2-arm1-GAAMCAACTGACCTAYWCTGCTAT-A (33arm2-X352 58) 147POAAGACATTATTCAGACTCC (1845 39) 148MBX2-arm1-AAGACATTATTCAGACTC-arm2-X3C (1845 39)
TABLE 2L1-specific sequences for inclusionin NASBA/PCR primers and probesPrimer/probeSEQ IDtypeSequence149P2/PCRAATGGCATTTGTTGGGGTAA 150P1/PCRX1-TCATATTCCTCCCCATGTC 151POTTGTTACTGTTGTTGATACTAC 152P2/PCRAATGGCATTTGTTGGSRHAA 153P1/PCRX1-TCATATTCCTCMMCATGDC 154POTTGTTACTGTTGTTGATACYAC 155POTTGTTACTGTTGTTGATACCAC 156P2/PCRAATGGCATTTGTTGGSIIAA 157P2/PCRAATGGCATTTGTTGGIIHAA 158P2/PCRAATGGCATTTGTTGGIRIAA 159P2/PCRAATGGCATTTGTTGGGGTAA 160P2/PCRAATGGCATTTGTTGGGGAAA 161P2/PCRAATGGCATTTGTTGGCATAA 162P2/PCRAATGGCATTTGTTGGGGCAA 163P2/PCRAATGGCATTTGTTGGCACAA 164P1/PCRX1-TCATATTCCTCMICATGIC 165P1/PCRX1-TCATATTCCTCAACATGIC 166P1/PCRX1-TCATATTCCTCIICATGTC 167P1/PCRX1-TCATATTCCTCIICATGGC 168P1/PCRX1-TCATATTCCTCIICATGAC 3′ 169P1/PCRX1-TCATATTCCTCIICATGCC 3′
Preferred primers suitable for use in detection of HPV L1 and E6 mRNA by NASBA are listed in the following tables. However, these are merely illustrative and it is not intended that the scope of the invention should be limited to these specific molecules.
In the following Tables the NASBA P2 primers (p2) include the sequence GATGCAAGGTCGCATATGAG (SEQ ID No. 170) at the 5′ end; the NASBA P1 primers (p1) include the sequence AATTCTAATACGACTCACTATAGGGAGAAGG (SEQ ID No. 172) at the 5′ end. Oligonucleotides suitable for use as probes are identified by “po”. The P2 primers generally contain HPV sequences from the positive strand, whereas the P1 primers generally contain HPV sequences from the negative strand. nt-refers to nucleotide position in the relevant HPV genomic sequence.
TABLE 3Preferred E6 NASBA primers and probesHPVPrimer nameSequenceTypentHAe6701p2GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC16116(SEQ ID 173)AGAAAGTTA HAe6701p1AATTCTAATACGACTCACTATAGGGAGAAGGACGG16368(SEQ ID 174)TTTGTTGTATTGCTGTTC HAe6702p2GATGCAAGGTCGCATATGAGCCACAGGAGCGACCC16116(SEQ ID 175)AGAAA HAe6702p1AATTCTAATACGACTCACTATAGGGAGAAGGGGTT16368(SEQ ID 176)TGTTGTATTGCTGTTC HPV16p1AATTCTAATACGACTCACTATAGGGAGAAGGATTC16258(SEQ ID 177)CCATCTCTATATACTA HAe6702Ap1AATTCTAATACGACTCACTATAGGGAGAAGGTCA16208(SEQ ID 178)CGTCGCAGTAACTGT HAe6702Bp1AATTCTAATACGACTCACTATAGGGAGAAGGTTG16191(SEQ ID 179)CTTGCAGTACACACA HAe6702Cp1AATTCTAATACGACTCACTATAGGGAGAAGGTGC16186(SEQ ID 180)AGIACACACATTCTA HAe6702Dp1AATTCTAATACGACTCACTATAGGGAGAAGGGCA16185(SEQ ID 181)GTACACACATTCTAA H16e6702Ap2GATGCAAGGTCGCATATGAGACAGTTATGCACAGA16142(SEQ ID 182)GCT H16e6702Bp2GATGCAAGGTCGCATATGAGATATTAGAATGTGTG16182(SEQ ID 183)TAC H16e6702Cp2GATGCAAGGTCGCATATGAGTTAGAATGTGTGTAC16185(SEQ ID 184)TGC H16e6702Dp2GATGCAAGGTCGCATATGAGGAATGTGTGTACTGC16188(SEQ ID 185)AAG H16e6702ApoACAGTTATGCACAGAGCT16142(SEQ ID 10) H16e6702BpoATATTAGAATGTGTGTAC16182(SEQ ID 11) H16e6702CpoTTAGAATGTGTGTACTGC16185(SEQ ID 12) H16e6702DpoGAATGTGTGTACTGCAAG16188(SEQ ID 13) HAe6701poCTTTGCTTTTCGGGATTTATGC16235(SEQ ID 18) HAe6702poTATGACTTTGCTTTTCGGGA16230(SEQ ID 19) HAe6702mb1X2-cgcatgTATGACTTTGCTTTTCGGGAcatgcg-16230(SEQ ID 186)X3 HAe6702mb2X2-ccagctTATGACTTTGCTTTTCGGGAagctgg-16230(SEQ ID 187)X3 HAe6702mb3X2-cacgcTATGACTTTGCTTTTCGGGAgcgtg-X316230(SEQ ID 188) H16e6702mb4X2-cgatcgTATGACTTTGCTTTTCGGGAcgatcg-16230(SEQ ID 189)X3 HAe6703p2GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG16656(SEQ ID 190)AAATAGTA HAe6703p1AATTCTAATACGACTCACTATAGGGAGAAGGGCAC16741(SEQ ID 191)AACCGAAGCGTAGAGTCACAC HAe6703poTGGACAAGCAGAACCGGACAGAGC16687(SEQ ID 23) HAe6704p2GATGCAAGGTCGCATATGAGCAGAGGAGGAGGATG16656(SEQ ID 192)AAATAGA HAe6704p1AATTCTAATACGACTCACTATAGGGAGAAGGGCAC16741(SEQ ID 193)AACCGAAGCGTAGAGTCA HAe6704poAGCAGAACCGGACAGAGCCCATTA16693(SEQ ID 26) H18e6701p2GATGCAAGGTCGCATATGAGACGATGAAATAGATG18702(SEQ ID 194)GAGTT H18e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGCACG18869(SEQ ID 195)GACACACAAAGGACAG Hl8e6701poAGCCGAACCACAACGTCACA18748(SEQ ID 29) H18e6702p2GATGCAAGGTCGCATATGAGGAAAACGATGAAATA18698(SEQ ID 196)GATGGAG H18e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGACAC18869(SEQ ID 197)CACGGACACACAAAGGACAG H18e6702poGAACCACAACGTCACACAATG18752(SEQ ID 32) H18e6702mb1X2-cgcatgGAACCACAACGTCACACAATGcatgcg-18752(SEQ ID 198)X3 H18e6702mb2X2-ccgtcgGAACCACAACGTCACACAATGcgacgg-18752(SEQ ID 199)X3 H18e6702mb3X2-cggaccGAACCACAACGTCACACAATGggtccg-18752(SEQ ID 200)X3 H18e6702mb4X2-cgatcgGAACCACAACGTCACACAATGcgatcg-18752(SEQ ID 201)X3 H18e6703p2GATGCAAGGTCGCATATGAGTTCCGGTTGACCTTC18651(SEQ ID 202)TATGT H18e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGGGTC18817(SEQ ID 203)GTCTGCTGAGCTTTCT H18e6704p2GATGCAAGGTCGCATATGAGGCAAGACATAGAAAT18179(SEQ ID 204)AACCTG H18e6704p1AATTCTAATACGACTCACTATAGGGAGAAGGACCC18379(SEQ ID 205)AGTGTTAGTTAGTT H18e6704poTGCAAGACAGTATTGGAACT18207(SEQ ID 38) H31e6701p2GATGCAAGGTCGCATATGAGGGAAATACCCTACGA31164(SEQ ID 206)TGAAC H31e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGGGAC31423(SEQ ID 207)ACAACGGTCTTTGACA H31e6701poATAGGGACGACACACCACACGGAG31268(SEQ ID 41) H31e6702p2GATGCAAGGTCGCATATGAGGGAAATACCCTACGA31164(SEQ ID 208)TGAACTA H31e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGCTGG31423(SEQ ID 209)ACACAACGGTCTTTGACA H31e6702poTAGGGACGACACACCACACGGA31269(SEQ ID 44) H31e6703p2GATGCAAGGTCGCATATGAGACTGACCTCCACTGT31617(SEQ ID 210)TATGA H31e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGTATC31766(SEQ ID 211)TACTTGTGTGCTCTGT H31e6703poGACAAGCAGAACCGGACACATC31687(SEQ ID 47) H31e6704p2GATGCAAGGTCGCATATGAGTGACCTCCACTGTTA31619(SEQ ID 212)TGAGCAATT H31e6704p1AATTCTAATACGACTCACTATAGGGAGAAGGTGCG31766(SEQ ID 213)AATATCTACTTGTGTGCTCT GT H31e6704poGGACAAGCAGAACCGGACACATCCAA31686(SEQ ID 50) H31e6704mb1X2-ccgaaggGGACAAGCAGAACCGGACACATCC31686(SEQ ID 214)AAccttcgg-X3 H31e6704mb2X2-ccgtcgGGACAAGCAGAACCGGACACATCCA31686(SEQ ID 215)Acgacgg-X3 H31e6704mb3X2-cacgtcgGGACAAGCAGAACCGGACACATCCAA31686(SEQ ID 216)cgacgtg-X3 H31e6704mb4X2-cgcagcGGACAAGCAGAACCGGACACATCCAA31686(SEQ ID 217)gctgcg-X3 H31e6704mb5X2-cgatcgGGACAAGCAGAACCGGACACATCCAA31686(SEQ ID 218)cgatcg-X3 H31e6705p2GATGCAAGGTCGCATATGAGACTGACCTCCACTGT31617(SEQ ID 219)TAT H31e6705p1AATTCTAATACGACTCACTATAGGGAGAAGGCACG31809(SEQ ID 220)ATTCCAAATGAGCCCAT H33e6701p2GATGCAAGGTCGCATATGAGTATCCTGAACCAACT33618(SEQ ID 221)GACCTAT H33e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGTTGA33763(SEQ ID 222)CACATAAACGAACTG H33e6701poCAGATGGACAAGCACAACC33694(SEQ ID 56) H33e6703p2GATGCAAGGTCGCATATGAGTCCTGAACCAACTGA33620(SEQ ID 223)CCTAT H33e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGCCCA33807(SEQ ID 224)TAAGTAGTTGCTGTAT H33e6703poGGACAAGCACAACCAGCCACAGC33699(SEQ ID 59) H33e6703mb1X2-ccaagcGGACAAGCACAACCAGCCACAGCgct33699(SEQ ID 225)tgg-X3 H33e6703mb2X2-ccaagcgGGACAAGCACAACCAGCCACAGC33699(SEQ ID 226)cgcttgg-X3 H33e6703mb3X2-cccagcGGACAAGCACAACCAGCCACAGCgct33699(SEQ ID 227)ggg-X3 H33e6703mb4X2-ccaaagcGGACAAGCACAACCAGCCACAGCg33699(SEQ ID 228)ctttgg-X3 H33e6703mb5X2-cctgcGGACAAGCACAACCAGCCACAGCgcagg-33699(SEQ ID 229)X3 H33e6703mb6X2-ccgatcgGGACAAGCACAACCAGCCACAGCcga33699(SEQ ID 230)tcg-X3 H33e6702p2GATGCAAGGTCGCATATGAGGACCTTTGTGTCCTC33431(SEQ ID 231)AAGAA H33e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGAGGT33618(SEQ ID 232)CAGTTGGTTCAGGATA H33e6702poAGAAACTGCACTGTGACGTGT33543(SEQ ID 63) H35e6701p2GATGCAAGGTCGCATATGAGATTACAGCGGAGTGA35217(SEQ ID 233)GGTAT H35e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGGTCT35442(SEQ ID 234)TTGCTTTTCAACTGGA H35e5601poATAGAGAAGGCCAGCCATAT35270(SEQ ID 66) H35e6702p2GATGCAAGGTCGCATATGAGTCAGAGGAGGAGGAA35655(SEQ ID 235)GATACTA H35e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGGATT35844(SEQ ID 236)ATGCTCTCTGTGAACA H35e6703p2GATGCAAGGTCGCATATGAGCCCGAGGCAACTGAC35610(SEQ ID 237)CTATA H35e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGGTCA35770(SEQ ID 238)ATGTGTGTGCTCTGTA H35e6702poGACAAGCAAAACCAGACACCTCCAA35692(SEQ ID 71) H35e6703poGACAAGCAAAACCAGACACC35692(SEQ ID 72) H52e6701p2GATGCAAGGTCGCATATGAGTTGTGTGAGGTGCTG52144(SEQ ID 239)GAAGAAT H52e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGCCCT52358(SEQ ID 240)CTCTTCTAATGTTT H52e6701poGTGCCTACGCTTTTTATCTA52296(SEQ ID 75) H52e6702p2GATGCAAGGTCGCATATGAGGTGCCTACGCTTTTT52296(SEQ ID 241)ATCTA H52e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGGGGG52507(SEQ ID 242)TCTCCAACACTCTGAACA H52e6702poTGCAAACAAGCGATTTCA52461(SEQ ID 78) H58e6701p2GATGCAAGGTCGCATATGAGTCAGGCGTTGGAGAC58157(SEQ ID 243)ATC H58e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGAGCA58301(SEQ ID 244)ATCGTAAGCACACT H58e6702p2GATGCAAGGTCGCATATGAGTCTGTGCATGAAATC58173(SEQ ID 245)GAA H58e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGAGCA58291(SEQ ID 246)CACTTTACATACTG H58e6701poTGAAATGCGTTGAATGCA58192(SEQ ID 83) H58e6702poTTGCAGCGATCTGAGGTATATG58218(SEQ ID 84) HBe6701p2GATGCAAGGTCGCATATGAGTACACTGCTGGACAAB (11)514(SEQ ID 247)CAT HBe6701p1AATTCTAATACGACTCACTATAGGGAGAAGGTCATB (11)619(SEQ ID 248)CTTCTGAGCTGTCT HBe6702p2GATGCAAGGTCGCATATGAGTACACTGCTGGACAAB (11)514(SEQ ID 249)CATGCA HBe6702p1AATTCTAATACGACTCACTATAGGGAGAAGGGTCAB (11)693(SEQ ID 250)CATCCACAGCAACAGGTCA HBe6701poGTAGGGTTACATTGCTATGAB (11)590(SEQ ID 89) HBe6702poGTAGGGTTACATTGCTATGAGCB (11)590(SEQ ID 90) HBe6703p2GATGCAAGGTCGCATATGAGTGACCTGTTGCTGTGB (11)693(SEQ ID 251)GATGTGA HBe6703p1AATTCTAATACGACTCACTATAGGGAGAAGGTACCB (11)832(SEQ ID 252)TGAATCGTCCGCCAT HBe6703poATWGTGTGTCCCATCTGCB (11)794(SEQ ID 93) HCe6701p2GATGCAAGGTCGCATATGAGCATGCCATAAATGTAC (18295(SEQ ID 253)TAGA39 45) HCe6701p1AATTCTAATACGACTCACTATAGGGAGAAGGCACCC (18408(SEQ ID 254)GCAGGCACCTTATTAA39 45 HCe6701poAGAATTAGAGAATTAAGAC (18324(SEQ ID 96)39 45 H39e6701p2GATGCAAGGTCGCATATGAGGCAGACGACCACTAC39210(SEQ ID 255)AGCAAA H39e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGACAC39344(SEQ ID 256)CGAGTCCGAGTAATA H39e6701poATAGGGACGGGGAACCACT39273(SEQ ID 99) H39e6702p2GATGCAAGGTCGCATATGAGTATTACTCGGACTCG39344(SEQ ID 257)GTGT H39e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGCTTG39558(SEQ ID 258)GGTTTCTCTTCGTGTTA H39e6702poGGACCACAAAACGGGAGGAC39531(SEQ ID 102) H39e6703p2GATGCAAGGTCGCATATGAGGAAATAGATGAACCC39703(SEQ ID 259)GACCA H39e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGGCAC39886(SEQ ID 260)ACCACGGACACACAAA H39e6703poTAGCCAGACGGGATGAACCACAGC39749(SEQ ID 105) HPV45p2GATGCAAGGTCGCATATGAGAACCATTGAACCCAG45430(SEQ ID 261)CAGAAA HPV45p1AATTCTAATACGACTCACTATAGGGAGAAGGTCTT45527(SEQ ID 262)TCTTGCCGTGCCTGGTCA HPV45poGTACCGAGGGCAGTGTAATA45500(SEQ ID 108) H45e6701p2GATGCAAGGTCGCATATGAGAACCATTGAACCCAG45430(SEQ ID 263)CAGAAA H45e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGTCTT45527(SEQ ID 264)TCTTGCCGTGCCTGGTCA H45e6702p2GATGCAAGGTCGCATATGAGGAAACCATTGAACCC45428(SEQ ID 265)AGCAGAAAA H45e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGTTGC45558(SEQ ID 266)TATACTTGTGTTTCCCTACG H45e6701poGTACCGAGGGCAGTGTAATA45500(SEQ ID 113) H45e6702poGGACAAACGAAGATTTCACA45467(SEQ ID 114) H45e6703p2GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT45656(SEQ ID 267)TACCAGCAAT H45e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGCACC45868(SEQ ID 268)ACGGACACACAAAGGACAAG H45e6704p2GATGCAAGGTCGCATATGAGCTGTTGACCTGTTGT45654(SEQ ID 269)GTTACGA H45e6704p1AATTCTAATACGACTCACTATAGGGAGAAGGCCAC45868(SEQ ID 270)GGACACACAAAGGACAAG H45e6705p2GATGCAAGGTCGCATATGAGGTTGACCTGTTGTGT45656(SEQ ID 271)TACGA H45e6705p1AATTCTAATACGACTCACTATAGGGAGAAGGACGG45868(SEQ ID 272)ACACACAAAGGACAAG H45e6703poGAGTCAGAGGAGGAAAACGATG45686(SEQ ID 121) H45e6704poAGGAAAACGATGAAGCAGATGGAGT45696(SEQ ID 122) H45e6705poACAACTACCAGCCCGACGAGCCGAA45730(SEQ ID 123) H51e6701p2GATGCAAGGTCGCATATGAGGGAGGAGGATGAAGT51658(SEQ ID 273)AGATA H51e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGGCCC51807(SEQ ID 274)ATTAACATCTGCTGTA H51e6702p2GATGCAAGGTCGCATATGAGAGAGGAGGAGGATGA51655(SEQ ID 275)AGTAGATA H51e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGACGG51829(SEQ ID 276)GCAAACCAGGCTTAGT H51e6701poGCAGGTGTTCAAGTGTAGTA51747(SEQ ID 128) H51e6702poTGGCAGTGGAAAGCAGTGGAGACA51771(SEQ ID 129) H56e6701p2GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA56519(SEQ ID 277)CAAACATCT H56e6701p1AATTCTAATACGACTCACTATAGGGAGAAGGTTCA56665(SEQ ID 278)TCCTCATCCTCATCCTCTGA H56e6702p2GATGCAAGGTCGCATATGAGTGGGGTGCTGGAGAC56520(SEQ ID 279)AAACATC H56e6702p1AATTCTAATACGACTCACTATAGGGAGAAGGCATC56665(SEQ ID 280)CTCATCCTCATCCTCTGA H56e6703p2GATGCAAGGTCGCATATGAGTTGGGGTGCTGGAGA56519(SEQ ID 281)CAAACAT H56e6703p1AATTCTAATACGACTCACTATAGGGAGAAGGCCAC56764(SEQ ID 282)AAACTTACACTCACAACA H56e6701poAAAGTACCAACGCTGCAAGACGT56581(SEQ ID 136) H56e6702poAGAACTAACACCTCAAACAGAAAT56610(SEQ ID 137) H56e6703poAGTACCAACGCTGCAAGACGTT56583(SEQ ID 138) H56e6703po1TTGGACAGCTCAGAGGATGAGG56656(SEQ ID 139) H56e6704p2GATGCAAGGTCGCATATGAGGATTTTCCTTATGCA56279(SEQ ID 283)GTGTG H56e6704p1AATTCTAATACGACTCACTATAGGGAGAAGGGACA56410(SEQ ID 284)TCTGTAGCACCTTATT H56e6704poGACTATTCAGTGTATGGAGC56348(SEQ ID 142) HPVAPO1ACAACTGAYCTMYACTGTTATGAA (16(SEQ ID 143)31 35) HPVApo1Amb1X2-cgcatgCAACTGAYCTMYACTGTTATGAcatgcA (16(SEQ ID 285)g-X331 35) HPVApo1Amb2X2-ccgtcgCAACTGAYCTMYACTGTTATGAcgaA (16(SEQ ID 286)cgg-X331 35) HPVApo1Amb3X2-ccacccCAACTGAYCTMYACTGTTATGAggA (16(SEQ ID 287)gtgg-X331 35) HPVApo1Amb4X2-cgatcgCAACTGAYCTMYACTGTTATGAcgaA (16(SEQ ID 288)tcg-X331 35) HPVAPO4AGAAMCAACTGACCTAYWCTGCTATA (33(SEQ ID 145)52 58) HPVAPO4Amb1X2-ccaagcGAAMCAACTGACCTAYWCTGCTATgcA (33(SEQ ID 289)ttgg-X352 58) HPVAPO4Amb2X2-ccaagccGAAMCAACTGACCTAYWCTGCTATA (33(SEQ ID 290)ggcttgg-X352 58) HPVAPO4Amb3X2-ccaagcgGAAMCAACTGACCTAYWCTGCTAA (33(SEQ ID 291)Tcgcttgg-X352 58) HPVAPO4Amb4X2-ccagcgGAAMCAACTGACCTAYWCTGCTATcgA (33(SEQ ID 292)ctgg-X352 58) HPVAPO4Amb5X2-cgatcgGAAMCAACTGACCTAYWCTGCTATcgA (33(SEQ ID 293)atcg-X352 58) HPVCPO4AAGACATTATTCAGACTCC (18(SEQ ID 147)45 39) HPVCPO4Amb1X2-ccaagcAAGACATTATTCAGACTCgcttgg-X3C (18(SEQ ID 294)45 39) HPVCPO4Amb2X2-cgcatgAAGACATTATTCAGACTCcatgcg-X3C (18(SEQ ID 295)45 39) HPVCPO4Amb3X2-cccagcAAGACATTATTCAGACTCgctggg-X3C (18(SEQ ID 296)45 39) HPVCPO4Amb4X2-cgatcgAAGACATTATTCAGACTCcgatcg-X3C (18(SEQ ID 297)45 39)Pairs of P1 and P2 primers having the same prefix (e.g. HAe6701p1 and HAe6701p2) are intended to be used in combination. However, other combinations may also be used, as summarised below for HPV types 16, 18, 31, 33 and 45.Suitable primer-pairs for amplification of HPV 16 E6 mRNA are as follows:    HAe6701p2 or HAe6702p2 (both nt 116) with HAe6701p1 or HAe6702p1 (both nt 368).    HAe6701p2 or HAe6702p2 (both nt 116) with HPV16p1(nt 258).    H16e6702Ap2 (nt 142), H16e6702 Bp2 (nt 182), H16e6702 Cp2 (nt 185) or H16e6702Dp2 (nt 188) with HAe6701p1 or HAe6702p1 (both nt 368).    HAe6701p2 or HAe6702p2 (both nt 116) with HAe6702Ap1 (nt 208), HAe6702 Bp1 (nt 191), HAe6702 Cp1 (nt 186) or HAe6702Dp1 (185). These combinations are suitable for amplification of all E6 splice variants.    HAe6703p2 or HAe6704p2 (both nt 656) with HAe6703p1 or HAe6704p1 (both nt 741). These combinations are suitable for amplification of all transcripts containing the E7 coding region (at least up to nt 741).The following primer-pairs are preferred for amplification of HPV 18 E6 mRNA:    H18e6701p2 (nt 702) or H18e6702p2 (nt 698) with H18e6701p1 or H18e6702p1 (both nt 869).    H18e6703p2 (nt 651) with H18e6703p1 (nt 817).    H18e6704p2 (nt 179) with H18e6704p1 (nt 379).The following primer-pairs are preferred for amplification of HPV 31 E6 mRNA:    H31e6701p2 or H31e6702p2 (both nt 164) with H31e6701p1 or H31e6702p1 (both nt 423).    H31e6703p2 (nt 617), H31e6704p2 (nt 619) or H31e6705p2 (nt 617) with H31e6703p1 (nt 766), H31e6704p1 (766) or H31e6705p1 (nt 809).The following primer-pairs are preferred for amplification of HPV 33 E6 mRNA:    H33e6701p2 (nt 618) or H33e6703p2 (nt 620) with H33e6701p1 (nt 763) or H33e6703p1 (nt 807).    H33e6702p2 (nt 431) with H33e6702p1 (nt 618).The following primer pair is preferred for amplification of HPV 45:    HPV45p2 (nt 430) with HPV45p1(nt 527)
TABLE 4E6 PCR primersHPVPrimer nameSequencetypentHAe6701PCR2(SEQ ID 1)CCACAGGAGCGACCCAGAAAGTTA16116 HAe6701PCR1(SEQ ID 2)ACGGTTTGTTGTATTGCTGTTC16368 HAe6702PCR2(SEQ ID 3)CCACAGGAGCGACCCAGAAA16116 HAe6702PCR1(SEQ ID 4)GGTTTGTTGTATTGCTGTTC16368 HAe6703PCR2(SEQ ID 21)CAGAGGAGGAGGATGAAATAGTA16656 HAe6703PCR1(SEQ ID 22)GCACAACCGAAGCGTAGAGTCACAC16741 HAe6704PCR2(SEQ ID 24)CAGAGGAGGAGGATGAAATAGA16656 HAe6704PCR1(SEQ ID 25)GCACAACCGAAGCGTAGAGTCA16741 H18e6701PCR2(SEQ ID 27)ACGATGAAATAGATGGAGTT18702 H18e6701PCR1(SEQ ID 28)CACGGACACACAAAGGACAG18869 H18e6702PCR2(SEQ ID 30)GAAAACGATGAAATAGATGGAG18698 H18e6702PCR1(SEQ ID 31)ACACCACGGACACACAAAGGACAG18869 H18e6703PCR2(SEQ ID 34)TTCCGGTTGACCTTCTATGT18651 H18e6703PCR1(SEQ ID 35)GGTCGTCTGCTGAGCTTTCT18817 H18e6704PCR2(SEQ ID 36)GCAAGACATAGAAATAACCTG18179 H18e6704PCR1(SEQ ID 37)ACCCAGTGTTAGTTAGTT18379 H31e6701PCR2(SEQ ID 39)GGAAATACCCTACGATGAAC31164 H31e6701PCR1(SEQ ID 40)GGACACAACGGTCTTTGACA31423 H31e6702PCR2(SEQ ID 42)GGAAATACCCTACGATGAACTA31164 H31e6702PCR1(SEQ ID 43)CTGGACACAACGGTCTTTGACA31423 H31e6703PCR2(SEQ ID 45)ACTGACCTCCACTGTTATGA31617 H31e6703PCR1(SEQ ID 46)TATCTACTTGTGTGCTCTGT31766 H31e6704PCR2(SEQ ID 48)TGACCTCCACTGTTATGAGCAATT31619 H31e6704PCR1(SEQ ID 49)TGCGAATATCTACTTGTGTGCTCTGT31766 H31e6705PCR2(SEQ ID 52)ACTGACCTCCACTGTTAT31617 H31e6705PCR1(SEQ ID 53)CACGATTCCAAATGAGCCCAT31809 H33e6701PCR2(SEQ ID 54)TATCCTGAACCAACTGACCTAT33618 H33e6701PCR1(SEQ ID 55)TTGACACATAAACGAACTG33763 H33e6703PCR2(SEQ ID 57)TCCTGAACCAACTGACCTAT33620 H33e6703PCR1(SEQ ID 58)CCCATAAGTAGTTGCTGTAT33807 H33e6702PCR2(SEQ ID 61)GACCTTTGTGTCCTCAAGAA33431 H33e6702PCR1(SEQ ID 62)AGGICAGTIGGITCAGGATA33618 H35e6701PCR2(SEQ ID 64)ATTACAGCGGAGTGAGGTAT35217 H35e6701PCR1(SEQ ID 65)GTCTTTGCTTTTCAACTGGA35442 H35e6702PCR2(SEQ ID 67)TCAGAGGAGGAGGAAGATACTA35655 H35e6702PCR1(SEQ ID 68)GATTATGCTCTCTGTGAACA35844 H35e6703PCR2(SEQ ID 69)CCCGAGGCAACTGACCTATA35610 H35e6703PCR1(SEQ ID 70)GTCAATGTGTGTGCTCTGTA35770 H52e6701PCR2(SEQ ID 73)TTGTGTGAGGTGCTGGAAGAAT52144 H52e6701PCR1(SEQ ID 74)CCCTCTCTTCTAATGTTT52358 H52e6702PCR2(SEQ ID 75)GTGCCTACGCTTTTTATCTA52296 H52e6702PCR1(SEQ ID 77)GGGGTCTCCAACACTCTGAACA52507 H58e6701PCR2(SEQ ID 79)TCAGGCGTTGGAGACATC58157 H58e6701PCR1(SEQ ID 80)AGCAATCGTAAGCACACT58301 H58e6702PCR2(SEQ ID 81)TCTGTGCATGAAATCGAA58173 H58e6702PCR1(SEQ ID 82)AGCACACTTTACATACTG58291 HBe6701PCR2(SEQ ID 85)TACACTGCTGGACAACATB (11)514 HBe6701PCR1(SEQ ID 86)TCATCTTCTGAGCTGTCTB (11)619 HBe6702PCR2(SEQ ID 87)TACACTGCTGGACAACATGCAB (11)514 HBe6702PCR1(SEQ ID 88)GTCACATCCACAGCAACAGGTCAB (11)693 HBe6703PCR2(SEQ ID 91)TGACCTGTTGCTGTGGATGTGAB (11)693 HBe6703PCR1(SEQ ID 92)TACCTGAATCGTCCGCCATB (11)832 HCe6701PCR2(SEQ ID 94)CATGCCATAAATGTATAGAC (1829539 45 HCe6701PCR1(SEQ ID 95)CACCGCAGGCACCTTATTAAC (1840839 45 H39e6701PCR2(SEQ ID 97)GCAGACGACCACTACAGCAAA39210 H39e6701PCR1(SEQ ID 98)ACACCGAGTCCGAGTAATA39344 H39e6702PCR2(SEQ ID 100)TATTACTCGGACTCGGTGT39344 H39e6702PCR1(SEQ ID 101)CTTGGGTTTCTCTTCGTGTTA39558 H39e6703PCR2(SEQ ID 103)GAAATAGATGAACCCGACCA39703 H39e6703PCR1(SEQ ID 104)GCACACCACGGACACACAAA39886 H45e6701PCR2(SEQ ID 106)AACCATTGAACCCAGCAGAAA45430 H45e6701PCR1(SEQ ID 107)TCTTTCTTGCCGTGCCTGGTCA45527 H45e6702PCR2(SEQ ID 111)GAAACCATTGAACCCAGCAGAAAA45428 H45e6702PCR1(SEQ ID 112)TTGCTATACTTGTGTTTCCCTACG45558 H45e6703PCR2(SEQ ID 115)GTTGACCTGTTGTGTTACCAGCAAT45656 H45e6703PCR1(SEQ ID 116)CACCACGGACACACAAAGGACAAG45868 H45e6704PCR2(SEQ ID 117)CTGTTGACCTGTTGTGTTACGA45654 H4Se6704PCR1(SEQ ID 118)CCACGGACACACAAAGGACAAG45868 H45e6705PCR2(SEQ ID 119)GTTGACCTGTTGTGTTACGA45656 H45e6705PCR1(SEQ ID 120)ACGGACACACAAAGGACAAG45868 H51e6701PCR2(SEQ ID 124)GGAGGAGGATGAAGTAGATA51658 H51e6701PCR1(SEQ ID 125)GCCCATTAACATCTGCTGTA51807 H51e6702PCR2(SEQ ID 126)AGAGGAGGAGGATGAAGTAGATA51655 H51e6702PCR1(SEQ ID 127)ACGGGCAAACCAGGCTTAGT51829 H56e6701PCR2(SEQ ID 130)TTGGGGTGCTGGAGACAAACATCT56519 H56e6701PCR1(SEQ ID 131)TTCATCCTCATCCTCATCCTCTGA56665 H56e6702PCR2(SEQ ID 132)TGGGGTGCTGGAGACAAACATC56520 H56e6702PCR1(SEQ ID 133)CATCCTCATCCTCATCCTCTGA56665 H56e6703PCR2(SEQ ID 134)TTGGGGTGCTGGAGACAAACAT56519 H56e6703PCR1(SEQ ID 135)CCACAAACTTACACTCACAACA56764 H56e6704PCR2(SEQ ID 140)GATTTTCCTTATGCAGTGTG56279 H56e6704PCR1(SEQ ID 141)GACATCTGTAGCACCTTATT56410Preferred PCR primer-pairs for HPV types 16, 18, 31 and 33 are analogous to the NASBA primer-pairs.
TABLE 5Preferred L1 NASBA primers and probesPrimernameSequenceOnc2A25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 298)GGGTAA 3′ Onc2A15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 299)TATTCCTCCCCATGTC 3′ Onc2PoA5′ TTGTTACTGTTGTTGATACTAC 3′(SEQ ID 151) Onc2B25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 300)GSRHAA 3′ Onc2B15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 301)TATTCCTCMMCATGDC 3′ Onc2PoB5′ TTGTTACTGTTGTTGATACYAC 3′(SEQ ID 154) Onc2PoC5′ TTGTTACTGTTGTTGATACCAC 3′(SEQ ID 155) Onc2C25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 302)GSIIAA 3′ Onc2D25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 303)GIIHAA 3′ Onc2E25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 304)GIRIAA 3′ Onc2F25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 305)GGGTAA 3′ Onc2G25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 306)GGGAAA 3′ Onc2H25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 307)GCATAA 3′ Onc2I25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 308)GGGCAA 3′ Onc2J25′ GATGCAAGGTCGCATATGAGAATGGCATTTGTTG(SEQ ID 309)GCACAA 3′ Onc2K15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 310)TATTCCTCMICATGIC 3′ Onc2L15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 311)TATTCCTCAACATGIC 3′ Onc2M15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 312)TATTCCTCIICATGTC 3′ Onc2N15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 313)TATTCCTCIICATGGC 3′ Onc2O15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 314)TATTCCTCIICATGAC 3′ Onc2P15′ AATTCTAATACGACTCACTATAGGGAGAAGGTCA(SEQ ID 315)TATTCCTCIICATGCC 3′
TABLE 6Preferred L1 PCR primersPrimer nameSequenceOnc2A1-PCR (SEQ ID 149)5′ AATGGCATTTGTTGGGGTAA 3′ Onc2A2-PCR (SEQ ID 150)5′ TCATATTCCTCCCCATGTC 3′ Onc2B1-PCR (SEQ ID 152)5′ AATGGCATTTGTTGGSRHAA 3′ Onc2B2-PCR (SEQ ID 153)5′ TCATATTCCTCMMCATGDC 3′ Onc2C1-PCR (SEQ ID 156)5′ AATGGCATTTGTTGGSIIAA 3′ Onc2D1-PCR (SEQ ID 157)5′ AATGGCATTTGTTGGIIHAA 3′ Onc2E1-PCR (SEQ ID 158)5′ AATGGCATTTGTTGGIRIAA 3′ Onc2F1-PCR (SEQ ID 159)5′ AATGGCATTTGTTGGGGTAA 3′ Onc2G1-PCR (SEQ ID 160)5′ AATGGCATTTGTTGGGGAAA 3′ Onc2H1-PCR (SEQ ID 161)5′ AATGGCATTTGTTGGCATAA 3′ Onc2I1-PCR (SEQ ID 162)5′ AATGGCATTTGTTGGGGCAA 3′ Onc2J1-PCR (SEQ ID 163)5′ AATGGCATTTGTTGGCACAA 3′ Onc2K2-PCR (SEQ ID 164)5′ TCATATTCCTCMICATGIC 3′ Onc2L2-PCR (SEQ ID 165)5′ TCATATTCCTCAACATGIC 3′ Onc2M2-PCR (SEQ ID 166)5′ TCATATTCCTCIICATGTC 3′ Onc2N2-PCR (SEQ ID 167)5′ TCATATTCCTCIICATGGC 3′ Onc2O2-PCR (SEQ ID 168)5′ TCATATTCCTCIICATGAC 3′ Onc2P2-PCR (SEQ ID 169)5′ TCATATTCCTCIICATGCC 3′
The HPV-specific sequences in SEQ ID NOs:149 and 150 (primers Onc2A2/Onc2A1-PCR and Onc2A1/Onc2A2-PCR) are identical to fragments of the HPV type 16 genomic sequence from position 6596-6615 (SEQ ID NO:149; Onc2A2/Onc2A1-PCR), and from position 6729 to 6747(SEQ ID NO:150; Onc2A1/Onc2A2-PCR).
The HPV-specific sequences SEQ ID NOs:152 and 153 (Onc2B2/Onc2B1-PCR and Onc2B1/Onc2B2-PCR) are variants of the above sequences, respectively, including several degenerate bases. Representations of the sequences of degenerate oligonucleotide molecules provided herein use the standard IUB code for mixed base sites: N=G, A, T, C; V=G, A, C; B=G, T, C; H=A, T, C; D=G, A, T; K=G, T; S=G, C; W=A, T; M=A, C; Y=C, T; R=A, G.
It is also possible to use variants of the HPV-specific sequences SEQ ID NO:152 (Onc2B2/Onc2B1-PCR) and SEQ ID NO:153 (Onc2B1/Onc2B2-PCR) wherein any two of nucleotides “SRH” towards the 3′ end of the sequence are replaced with inosine (I), as follows:
5′ AATGGCATTTGTTGGIIHAA 3′(SEQ ID 157) 5′ AATGGCATTTGTTGGSIIAA 3′(SEQ ID 156) 5′ AATGGCATTTGTTGGIRIAA 3′(SEQ ID 158)
The HPV-specific sequences SEQ ID NOs: 156-163 (present in primers Onc2C2, Onc2D2, Onc2E2, Onc2F2, Onc2G2, Onc2H2, Onc212, Onc2J2, Onc2C1-PCR, Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR, Onc2H1-PCR, Onc211-PCR and Onc2J1-PCR) are variants based on the HPV-specific sequence SEQ ID NO:152 (Onc2B2/Onc2B1-PCR), whereas the HPV-specific sequences SEQ ID NOs: 164-169 (present in primers Onc2K1, Onc2L1, Onc2M1, Onc2N1, Onc201, Onc2P1, Onc2K2-PCR, Onc2L2-PCR, Onc2M2-PCR, Onc2N2-PCR, Onc202-PCR and Onc2P2-PCR are variants based on the HPV-specific sequence SEQ ID NO:153 (Onc2B1/Onc2B2-PCR). These variants include degenerate bases and also inosine (I) residues. This sequence variation enables oligonucleotides incorporating the variant sequences to bind to multiple HPV types. Inosine bases do not interfere with hybridization and so may be included at sites of variation between HPV types in order to construct a “consensus” primer able to bind to multiple HPV types.
Any one or more of primers Onc2A2, Onc2B2, Onc2C2, Onc2D2, Onc2E2, Onc2F2, Onc2G2, Onc2H2, Onc212 and Onc2J2, may be used in combination with any one or more of primers Onc2A1, Onc2B1, Onc2K1, Onc2L1, Onc2M1, Onc2N1, Onc201 and Onc2P1, for NASBA amplification of HPV L1 mRNA.
Any one or more of primers Onc2A1-PCR, Onc2B1-PCR, Onc2C1-PCR, Onc2D1-PCR, Onc2E1-PCR, Onc2F1-PCR, Onc2G1-PCR, Onc2H1-PCR, Onc2I1-PCR and Onc2J1-PCR, may be used in combination with any one or more of primers Onc2A2-PCR, Onc2B2-PCR, Onc2K2-PCR, Onc2L2-PCR, Onc2M2-PCR, Onc2N2-PCR, Onc202-PCR and Onc2P2-PCR for PCR amplification of HPV L1 mRNA.